Discovering Conformational Sub-States Relevant to Protein Function

PubMed Central

Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

2011-01-01

Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

Effects of Limited Hydrolysis and High-Pressure Homogenization on Functional Properties of Oyster Protein Isolates.

PubMed

Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Du, Ming

2018-03-22

In this study, the effects of limited hydrolysis and/or high-pressure homogenization (HPH) treatment in acid conditions on the functional properties of oyster protein isolates (OPI) were studied. Protein solubility, surface hydrophobicity, particle size distribution, zeta potential, foaming, and emulsifying properties were evaluated. The results showed that acid treatment led to the dissociation and unfolding of OPI. Subsequent treatment such as limited proteolysis, HPH, and their combination remarkably improved the functional properties of OPI. Acid treatment produced flexible aggregates, as well as reduced particle size and solubility. On the contrary, limited hydrolysis increased the solubility of OPI. Furthermore, HPH enhanced the effectiveness of the above treatments. The emulsifying and foaming properties of acid- or hydrolysis-treated OPI significantly improved. In conclusion, a combination of acid treatment, limited proteolysis, and HPH improved the functional properties of OPI. The improvements in the functional properties of OPI could potentiate the use of oyster protein and its hydrolysates in the food industry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deisenhofer, J.; Michel, H.

The history and methods of membrane protein crystallization are described. The solution of the structure of the photosynthetic reaction center from the bacterium Rhodopseudomonas viridis is described, and the structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Conclusions about the structure of the photosystem II reaction center from plants are drawn, and aspects of membrane protein structure are discussed. 68 refs., 15 figs., 2 tabs.

Prediction of scaffold proteins based on protein interaction and domain architectures.

PubMed

Oh, Kimin; Yi, Gwan-Su

2016-07-28

Scaffold proteins are known for being crucial regulators of various cellular functions by assembling multiple proteins involved in signaling and metabolic pathways. Identification of scaffold proteins and the study of their molecular mechanisms can open a new aspect of cellular systemic regulation and the results can be applied in the field of medicine and engineering. Despite being highlighted as the regulatory roles of dozens of scaffold proteins, there was only one known computational approach carried out so far to find scaffold proteins from interactomes. However, there were limitations in finding diverse types of scaffold proteins because their criteria were restricted to the classical scaffold proteins. In this paper, we will suggest a systematic approach to predict massive scaffold proteins from interactomes and to characterize the roles of scaffold proteins comprehensively. From a total of 10,419 basic scaffold protein candidates in protein interactomes, we classified them into three classes according to the structural evidences for scaffolding, such as domain architectures, domain interactions and protein complexes. Finally, we could define 2716 highly reliable scaffold protein candidates and their characterized functional features. To assess the accuracy of our prediction, the gold standard positive and negative data sets were constructed. We prepared 158 gold standard positive data and 844 gold standard negative data based on the functional information from Gene Ontology consortium. The precision, sensitivity and specificity of our testing was 80.3, 51.0, and 98.5 % respectively. Through the function enrichment analysis of highly reliable scaffold proteins, we could confirm the significantly enriched functions that are related to scaffold protein binding. We also identified functional association between scaffold proteins and their recruited proteins. Furthermore, we checked that the disease association of scaffold proteins is higher than kinases. In conclusion, we could predict larger volume of scaffold proteins and analyzed their functional characteristics. Deeper understandings about the roles of scaffold proteins from this study will provide a higher opportunity to find therapeutic or engineering applications of scaffold proteins using their functional characteristics.

Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

PubMed

Busk, Peter Kamp; Lange, Lene

2013-06-01

Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.

Predicting Functions of Proteins in Mouse Based on Weighted Protein-Protein Interaction Network and Protein Hybrid Properties

PubMed Central

Shi, Xiaohe; Lu, Wen-Cong; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

Background With the huge amount of uncharacterized protein sequences generated in the post-genomic age, it is highly desirable to develop effective computational methods for quickly and accurately predicting their functions. The information thus obtained would be very useful for both basic research and drug development in a timely manner. Methodology/Principal Findings Although many efforts have been made in this regard, most of them were based on either sequence similarity or protein-protein interaction (PPI) information. However, the former often fails to work if a query protein has no or very little sequence similarity to any function-known proteins, while the latter had similar problem if the relevant PPI information is not available. In view of this, a new approach is proposed by hybridizing the PPI information and the biochemical/physicochemical features of protein sequences. The overall first-order success rates by the new predictor for the functions of mouse proteins on training set and test set were 69.1% and 70.2%, respectively, and the success rate covered by the results of the top-4 order from a total of 24 orders was 65.2%. Conclusions/Significance The results indicate that the new approach is quite promising that may open a new avenue or direction for addressing the difficult and complicated problem. PMID:21283518

The speciation of the proteome

PubMed Central

Jungblut, Peter R; Holzhütter, Hermann G; Apweiler, Rolf; Schlüter, Hartmut

2008-01-01

Introduction In proteomics a paradox situation developed in the last years. At one side it is basic knowledge that proteins are post-translationally modified and occur in different isoforms. At the other side the protein expression concept disclaims post-translational modifications by connecting protein names directly with function. Discussion Optimal proteome coverage is today reached by bottom-up liquid chromatography/mass spectrometry. But quantification at the peptide level in shotgun or bottom-up approaches by liquid chromatography and mass spectrometry is completely ignoring that a special peptide may exist in an unmodified form and in several-fold modified forms. The acceptance of the protein species concept is a basic prerequisite for meaningful quantitative analyses in functional proteomics. In discovery approaches only top-down analyses, separating the protein species before digestion, identification and quantification by two-dimensional gel electrophoresis or protein liquid chromatography, allow the correlation between changes of a biological situation and function. Conclusion To obtain biological relevant information kinetics and systems biology have to be performed at the protein species level, which is the major challenge in proteomics today. PMID:18638390

[Analysis of virulence factors of Porphyromonas endodontalis based on comparative proteomics technique].

PubMed

Li, H; Ji, H; Wu, S S; Hou, B X

2016-12-09

Objective: To analyze the protein expression profile and the potential virulence factors of Porphyromonas endodontalis (Pe) via comparison with that of two strains of Porphyromonas gingivalis (Pg) with high and low virulences, respectively. Methods: Whole cell comparative proteomics of Pe ATCC35406 was examined and compared with that of high virulent strain Pg W83 andlow virulent strain Pg ATCC33277, respectively. Isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) were adopted to identify and quantitate the proteins of Pe and two strains of Pg with various virulences by using the methods of isotopically labeled peptides, mass spectrometric detection and bioinformatics analysis. The biological functions of similar proteins expressed by Pe ATCC35406 and two strains of Pg were quantified and analyzed. Results: Totally 1 210 proteins were identified while Pe compared with Pg W83. There were 130 proteins (10.74% of the total proteins) expressed similarly, including 89 known functional proteins and 41 proteins of unknown functions. Totally 1 223 proteins were identified when Pe compared with Pg ATCC33277. There were 110 proteins (8.99% of the total proteins) expressed similarly, including 72 known functional proteins and 38 proteins of unknown functions. The similarly expressed proteins in Pe and Pg strains with various virulences mainly focused on catalytic activity and binding function, including recombination activation gene (RagA), lipoprotein, chaperonin Dnak, Clp family proteins (ClpC and ClpX) and various iron-binding proteins. They were involved in metabolism and cellular processes. In addition, the type and number of similar virulence proteins between Pe and high virulence Pg were higher than those between Pe and low virulence Pg. Conclusions: Lipoprotein, oxygen resistance protein, iron binding protein were probably the potential virulence factors of Pe ATCC35406. It was speculated that pathogenicity of Pe was more similar to high virulence Pg than that to low virulence strain.

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

PubMed Central

2009-01-01

Background Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. Results To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Conclusion Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences. PMID:19821996

Classification of protein quaternary structure by functional domain composition

PubMed Central

Yu, Xiaojing; Wang, Chuan; Li, Yixue

2006-01-01

Background The number and the arrangement of subunits that form a protein are referred to as quaternary structure. Quaternary structure is an important protein attribute that is closely related to its function. Proteins with quaternary structure are called oligomeric proteins. Oligomeric proteins are involved in various biological processes, such as metabolism, signal transduction, and chromosome replication. Thus, it is highly desirable to develop some computational methods to automatically classify the quaternary structure of proteins from their sequences. Results To explore this problem, we adopted an approach based on the functional domain composition of proteins. Every protein was represented by a vector calculated from the domains in the PFAM database. The nearest neighbor algorithm (NNA) was used for classifying the quaternary structure of proteins from this information. The jackknife cross-validation test was performed on the non-redundant protein dataset in which the sequence identity was less than 25%. The overall success rate obtained is 75.17%. Additionally, to demonstrate the effectiveness of this method, we predicted the proteins in an independent dataset and achieved an overall success rate of 84.11% Conclusion Compared with the amino acid composition method and Blast, the results indicate that the domain composition approach may be a more effective and promising high-throughput method in dealing with this complicated problem in bioinformatics. PMID:16584572

NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

PubMed Central

Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

2006-01-01

Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

Insulator function and topological domain border strength scale with architectural protein occupancy

PubMed Central

2014-01-01

Background Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. Results By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. Conclusions We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains. PMID:24981874

Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs

PubMed Central

2011-01-01

Background One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Results Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Conclusions Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins. PMID:21689388

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.

PubMed

Ye, Junqiang; Beetz, Nadine; O'Keeffe, Sean; Tapia, Juan Carlos; Macpherson, Lindsey; Chen, Weisheng V; Bassel-Duby, Rhonda; Olson, Eric N; Maniatis, Tom

2015-06-09

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.

Functional and Genomic Features of Human Genes Mutated in Neuropsychiatric Disorders

PubMed Central

Forero, Diego A.; Prada, Carlos F.; Perry, George

2016-01-01

Background: In recent years, a large number of studies around the world have led to the identification of causal genes for hereditary types of common and rare neurological and psychiatric disorders. Objective: To explore the functional and genomic features of known human genes mutated in neuropsychiatric disorders. Methods: A systematic search was used to develop a comprehensive catalog of genes mutated in neuropsychiatric disorders (NPD). Functional enrichment and protein-protein interaction analyses were carried out. A false discovery rate approach was used for correction for multiple testing. Results: We found several functional categories that are enriched among NPD genes, such as gene ontologies, protein domains, tissue expression, signaling pathways and regulation by brain-expressed miRNAs and transcription factors. Sixty six of those NPD genes are known to be druggable. Several topographic parameters of protein-protein interaction networks and the degree of conservation between orthologous genes were identified as significant among NPD genes. Conclusion: These results represent one of the first analyses of enrichment of functional categories of genes known to harbor mutations for NPD. These findings could be useful for a future creation of computational tools for prioritization of novel candidate genes for NPD. PMID:27990183

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila.

PubMed

Barton, Lacy J; Wilmington, Shameika R; Martin, Melinda J; Skopec, Hannah M; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2014-06-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. Copyright © 2014 by the Genetics Society of America.

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

PubMed Central

Barton, Lacy J.; Wilmington, Shameika R.; Martin, Melinda J.; Skopec, Hannah M.; Lovander, Kaylee E.; Pinto, Belinda S.; Geyer, Pamela K.

2014-01-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. PMID:24700158

Modular architecture of protein structures and allosteric communications: potential implications for signaling proteins and regulatory linkages

PubMed Central

del Sol, Antonio; Araúzo-Bravo, Marcos J; Amoros, Dolors; Nussinov, Ruth

2007-01-01

Background Allosteric communications are vital for cellular signaling. Here we explore a relationship between protein architectural organization and shortcuts in signaling pathways. Results We show that protein domains consist of modules interconnected by residues that mediate signaling through the shortest pathways. These mediating residues tend to be located at the inter-modular boundaries, which are more rigid and display a larger number of long-range interactions than intra-modular regions. The inter-modular boundaries contain most of the residues centrally conserved in the protein fold, which may be crucial for information transfer between amino acids. Our approach to modular decomposition relies on a representation of protein structures as residue-interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for allosteric communications. The modular decomposition of 100 multi-domain protein structures indicates that modules constitute the building blocks of domains. The analysis of 13 allosteric proteins revealed that modules characterize experimentally identified functional regions. Based on the study of an additional functionally annotated dataset of 115 proteins, we propose that high-modularity modules include functional sites and are the basic functional units. We provide examples (the Gαs subunit and P450 cytochromes) to illustrate that the modular architecture of active sites is linked to their functional specialization. Conclusion Our method decomposes protein structures into modules, allowing the study of signal transmission between functional sites. A modular configuration might be advantageous: it allows signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulation of inter-modular linkages. PMID:17531094

The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity

PubMed Central

Stein, Matthias; Pilli, Manohar; Bernauer, Sabine; Habermann, Bianca H.; Zerial, Marino; Wade, Rebecca C.

2012-01-01

Background Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood. Methodology/Principal Findings Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics. Conclusions/Significance We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity. PMID:22523562

In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

PubMed Central

Lalucque, Hervé; Silar, Philippe

2000-01-01

Background To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. Results Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. Conclusion Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division. PMID:11112985

Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets*

PubMed Central

Yu, Xueping; Ivanic, Joseph; Memišević, Vesna; Wallqvist, Anders; Reifman, Jaques

2011-01-01

We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not necessarily imply that one detection methodology was better or worse, but rather that, to a large extent, the insights reflected the methodological biases themselves. Consequently, interpreting the protein interaction data within their experimental or cellular context provided the best avenue for overcoming biases and inferring biological knowledge. PMID:21876202

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

PubMed Central

Fay, Allison; Meyer, Pablo; Dworkin, Jonathan

2010-01-01

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the later steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a non-essential, sporulation-specific homolog of the well-conserved and essential SEDS proteins, and SpoVD, a non-essential class B penicillin binding protein (PBP). We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and Fluorescence Resonance Energy Transfer experiments indicated that SpoVE and SpoVD interact and co-affinity purification in E. coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of a SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in a loss of function of the entire fusion that was not complemented by a wild type protein. Thus, SpoVE has a direct and functional interaction with SpoVD and this conclusion will facilitate understanding the essential function SpoVE and related SEDS proteins such as FtsW and RodA play in bacterial growth and division. PMID:20417640

News from the protein mutability landscape.

PubMed

Hecht, Maximilian; Bromberg, Yana; Rost, Burkhard

2013-11-01

Some mutations of protein residues matter more than others, and these are often conserved evolutionarily. The explosion of deep sequencing and genotyping increasingly requires the distinction between effect and neutral variants. The simplest approach predicts all mutations of conserved residues to have an effect; however, this works poorly, at best. Many computational tools that are optimized to predict the impact of point mutations provide more detail. Here, we expand the perspective from the view of single variants to the level of sketching the entire mutability landscape. This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. We review some of the powerful conclusions about protein function, stability and their robustness to mutation that can be drawn from such an analysis. Large-scale experimental and computational mutagenesis experiments are increasingly furthering our understanding of protein function and of the genotype-phenotype associations. We also discuss how these can be used to improve predictions of protein function and pathogenicity of missense variants. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

PubMed Central

2012-01-01

Background The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. Results Our findings demonstrate that a miRNA’s functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. Conclusions We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment. PMID:22929553

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins.

PubMed

Revenu, Céline; Ubelmann, Florent; Hurbain, Ilse; El-Marjou, Fatima; Dingli, Florent; Loew, Damarys; Delacour, Delphine; Gilet, Jules; Brot-Laroche, Edith; Rivero, Francisco; Louvard, Daniel; Robine, Sylvie

2012-01-01

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.

Murine colon proteome and characterization of the protein pathways

PubMed Central

2012-01-01

Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer) rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse) whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR < 2) with comprehensive insight on its peptide properties, cellular and subcellular localization, functional network GO annotation analysis, and its relative abundances. The presented dataset includes wide spectra of pI and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9) in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2), Glutathione S-transferase (Gstp1) in prostate cancer, and Cell division control protein (Cdc42), Ras-related protein (Rac1,2) in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI) provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well. PMID:22929016

RRW: repeated random walks on genome-scale protein networks for local cluster discovery

PubMed Central

Macropol, Kathy; Can, Tolga; Singh, Ambuj K

2009-01-01

Background We propose an efficient and biologically sensitive algorithm based on repeated random walks (RRW) for discovering functional modules, e.g., complexes and pathways, within large-scale protein networks. Compared to existing cluster identification techniques, RRW implicitly makes use of network topology, edge weights, and long range interactions between proteins. Results We apply the proposed technique on a functional network of yeast genes and accurately identify statistically significant clusters of proteins. We validate the biological significance of the results using known complexes in the MIPS complex catalogue database and well-characterized biological processes. We find that 90% of the created clusters have the majority of their catalogued proteins belonging to the same MIPS complex, and about 80% have the majority of their proteins involved in the same biological process. We compare our method to various other clustering techniques, such as the Markov Clustering Algorithm (MCL), and find a significant improvement in the RRW clusters' precision and accuracy values. Conclusion RRW, which is a technique that exploits the topology of the network, is more precise and robust in finding local clusters. In addition, it has the added flexibility of being able to find multi-functional proteins by allowing overlapping clusters. PMID:19740439

Evolution and functional divergence of the anoctamin family of membrane proteins

PubMed Central

2010-01-01

Background The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family. Results Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins. Conclusions Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family. PMID:20964844

A domain-centric solution to functional genomics via dcGO Predictor

PubMed Central

2013-01-01

Background Computational/manual annotations of protein functions are one of the first routes to making sense of a newly sequenced genome. Protein domain predictions form an essential part of this annotation process. This is due to the natural modularity of proteins with domains as structural, evolutionary and functional units. Sometimes two, three, or more adjacent domains (called supra-domains) are the operational unit responsible for a function, e.g. via a binding site at the interface. These supra-domains have contributed to functional diversification in higher organisms. Traditionally functional ontologies have been applied to individual proteins, rather than families of related domains and supra-domains. We expect, however, to some extent functional signals can be carried by protein domains and supra-domains, and consequently used in function prediction and functional genomics. Results Here we present a domain-centric Gene Ontology (dcGO) perspective. We generalize a framework for automatically inferring ontological terms associated with domains and supra-domains from full-length sequence annotations. This general framework has been applied specifically to primary protein-level annotations from UniProtKB-GOA, generating GO term associations with SCOP domains and supra-domains. The resulting 'dcGO Predictor', can be used to provide functional annotation to protein sequences. The functional annotation of sequences in the Critical Assessment of Function Annotation (CAFA) has been used as a valuable opportunity to validate our method and to be assessed by the community. The functional annotation of all completely sequenced genomes has demonstrated the potential for domain-centric GO enrichment analysis to yield functional insights into newly sequenced or yet-to-be-annotated genomes. This generalized framework we have presented has also been applied to other domain classifications such as InterPro and Pfam, and other ontologies such as mammalian phenotype and disease ontology. The dcGO and its predictor are available at http://supfam.org/SUPERFAMILY/dcGO including an enrichment analysis tool. Conclusions As functional units, domains offer a unique perspective on function prediction regardless of whether proteins are multi-domain or single-domain. The 'dcGO Predictor' holds great promise for contributing to a domain-centric functional understanding of genomes in the next generation sequencing era. PMID:23514627

Improved Function With Enhanced Protein Intake per Meal: A Pilot Study of Weight Reduction in Frail, Obese Older Adults

PubMed Central

Pieper, Carl F.; Orenduff, Melissa C.; McDonald, Shelley R.; McClure, Luisa B.; Zhou, Run; Payne, Martha E.; Bales, Connie W.

2016-01-01

Abstract Background: Obesity is a significant cause of functional limitations in older adults; yet, concerns that weight reduction could diminish muscle along with fat mass have impeded progress toward an intervention. Meal-based enhancement of protein intake could protect function and/or lean mass but has not been studied during geriatric obesity reduction. Methods: In this 6-month randomized controlled trial, 67 obese (body mass index ≥30kg/m2) older (≥60 years) adults with a Short Physical Performance Battery score of 4–10 were randomly assigned to a traditional (Control) weight loss regimen or one with higher protein intake (>30g) at each meal (Protein). All participants were prescribed a hypo-caloric diet, and weighed and provided dietary guidance weekly. Physical function (Short Physical Performance Battery) and lean mass (BOD POD), along with secondary measures, were assessed at 0, 3, and 6 months. Results: At the 6-month endpoint, there was significant (p < .001) weight loss in both the Control (−7.5±6.2kg) and Protein (−8.7±7.4kg) groups. Both groups also improved function but the increase in the Protein (+2.4±1.7 units; p < .001) was greater than in the Control (+0.9±1.7 units; p < .01) group (p = .02). Conclusion: Obese, functionally limited older adults undergoing a 6-month weight loss intervention with a meal-based enhancement of protein quantity and quality lost similar amounts of weight but had greater functional improvements relative to the Control group. If confirmed, this dietary approach could have important implications for improving the functional status of this vulnerable population (ClinicalTrials.gov identifier: NCT01715753). PMID:26786203

The protein interaction map of bacteriophage lambda

PubMed Central

2011-01-01

Background Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. PMID:21943085

AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration.

PubMed

Herdegen, T; Waetzig, V

2001-04-30

Jun and Fos proteins are induced and activated following most physiological and pathophysiological stimuli in the brain. Only few data allow conclusions about distinct functions of AP-1 proteins in neurodegeneration and neuroregeneration, and these functions mainly refer to c-Jun and its activation by JNKs. Apoptotic functions of activated c-Jun affect hippocampal, nigral and primary cultured neurons following excitotoxic stimulation and destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs might exert neuroprotection by subsequent omission of c-Jun activation. Besides endogenous neuronal functions, the c-Jun/AP-1 proteins can damage the nervous system by upregulation of harmful programs in non-neuronal cells (e.g. microglia) with release of neurodegenerative molecules. In contrast, the differentiation with neurite extension and maturation of neural cells in vitro indicate physiological and potentially neuroprotective functions of c-Jun and JNKs including sensoring for alterations in the cytoskeleton. This review summarizes the multiple molecular interfunctions which are involved in the shift from the physiological role to degenerative effects of the Jun/JNK-axis such as cell type-specific expression and intracellular localization of scaffold proteins and upstream activators, antagonistic phosphatases, interaction with other kinase systems, or the activation of transcription factors competing for binding to JNK proteins and AP-1 DNA elements.

Enhanced Functional Recombinant Factor VII Production by HEK 293 Cells Stably Transfected with VKORC1 where the Gamma-Carboxylase Inhibitor Calumenin is Stably Suppressed by shRNA Transfection

PubMed Central

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Introduction Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo γ-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational γ carboxylation, the recovery of fully γ-carboxylated and functional proteins is low. Materials and Methods In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K γ-carboxylase cofactor and in addition stably silenced the γ-carboxylase inhibitory protein calumenin. Results and Conclusions Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C. PMID:18177690

DWARF – a data warehouse system for analyzing protein families

PubMed Central

Fischer, Markus; Thai, Quan K; Grieb, Melanie; Pleiss, Jürgen

2006-01-01

Background The emerging field of integrative bioinformatics provides the tools to organize and systematically analyze vast amounts of highly diverse biological data and thus allows to gain a novel understanding of complex biological systems. The data warehouse DWARF applies integrative bioinformatics approaches to the analysis of large protein families. Description The data warehouse system DWARF integrates data on sequence, structure, and functional annotation for protein fold families. The underlying relational data model consists of three major sections representing entities related to the protein (biochemical function, source organism, classification to homologous families and superfamilies), the protein sequence (position-specific annotation, mutant information), and the protein structure (secondary structure information, superimposed tertiary structure). Tools for extracting, transforming and loading data from public available resources (ExPDB, GenBank, DSSP) are provided to populate the database. The data can be accessed by an interface for searching and browsing, and by analysis tools that operate on annotation, sequence, or structure. We applied DWARF to the family of α/β-hydrolases to host the Lipase Engineering database. Release 2.3 contains 6138 sequences and 167 experimentally determined protein structures, which are assigned to 37 superfamilies 103 homologous families. Conclusion DWARF has been designed for constructing databases of large structurally related protein families and for evaluating their sequence-structure-function relationships by a systematic analysis of sequence, structure and functional annotation. It has been applied to predict biochemical properties from sequence, and serves as a valuable tool for protein engineering. PMID:17094801

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

PubMed Central

Wilson, Robert L.; Frisz, Jessica F.; Hanafin, William P.; Carpenter, Kevin J.; Hutcheon, Ian D.; Weber, Peter K.; Kraft, Mary L.

2014-01-01

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein’s activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests of whether specific proteins co-localize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein co-localization with specific lipid species. PMID:22284327

HEPATIC FUNCTION AFTER GENETICALLY-ENGINEERED PIG LIVER TRANSPLANTATION IN BABOONS

PubMed Central

Ekser, Burcin; Echeverri, Gabriel J.; Hassett, Andrea Cortese; Yazer, Mark H.; Long, Cassandra; Meyer, Michael; Ezzelarab, Mohamed; Lin, Chih Che; Hara, Hidetaka; van der Windt, Dirk J.; Dons, Eefje M.; Phelps, Carol; Ayares, David; Cooper, David K.C.; Gridelli, Bruno

2010-01-01

Background If ‘bridging’ to allotransplantation is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. Methods We have studied hepatic function in baboons after transplantation of livers from α1,3-galactosyltransferase gene-knockout (GTKO,n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46,n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In 4 baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. Results Recipient baboons died or were euthanized after 4-7 days following internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. Conclusions After the transplantation of genetically-engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near-normal; (2) there was evidence for production of pig proteins, including coagulation factors, and (3) these appeared to function adequately in baboons, though inter-species compatibility of such proteins remains to be confirmed. PMID:20606605

Identification and molecular characterisation of a homozygous missense mutation in the ADAMTS10 gene in a patient with Weill-Marchesani syndrome.

PubMed

Steinkellner, Hannes; Etzler, Julia; Gogoll, Laura; Neesen, Jürgen; Stifter, Eva; Brandau, Oliver; Laccone, Franco

2015-09-01

Weill-Marchesani syndrome is a rare disorder of the connective tissue. Functional variants in ADAMTS10 are associated with Weill-Marchesani syndrome-1. We identified a homozygous missense mutation, c.41T>A, of the ADAMTS10 gene in a 19-year-old female with typical symptoms of WMS1: proportionate short stature, brachydactyly, joint stiffness, and microspherophakia. The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence. Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum. A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Jinmei; Wang Xuefeng; Xi Zhiqin

2006-10-06

Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in themore » temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.« less

Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

DOE PAGES

Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; ...

2014-12-18

Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

[Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig].

PubMed

Luo, X; Wang, J Y; Zhang, F L; Xia, Y

2018-01-07

Objective: To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function. Methods: Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions. Results: The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein. Conclusion: EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

Protein Contribution to Plant Salinity Response and Tolerance Acquisition

PubMed Central

Kosová, Klára; Prášil, Ilja T.; Vítámvás, Pavel

2013-01-01

The review is focused on plant proteome response to salinity with respect to physiological aspects of plant salt stress response. The attention is paid to both osmotic and ionic effects of salinity stress on plants with respect to several protein functional groups. Therefore, the role of individual proteins involved in signalling, changes in gene expression, protein biosynthesis and degradation and the resulting changes in protein relative abundance in proteins involved in energy metabolism, redox metabolism, stressand defence-related proteins, osmolyte metabolism, phytohormone, lipid and secondary metabolism, mechanical stress-related proteins as well as protein posttranslational modifications are discussed. Differences between salt-sensitive (glycophytes) and salt-tolerant (halophytes) plants are analysed with respect to differential salinity tolerance. In conclusion, contribution of proteomic studies to understanding plant salinity tolerance is summarised and discussed. PMID:23531537

Query3d: a new method for high-throughput analysis of functional residues in protein structures

PubMed Central

Ausiello, Gabriele; Via, Allegra; Helmer-Citterich, Manuela

2005-01-01

Background The identification of local similarities between two protein structures can provide clues of a common function. Many different methods exist for searching for similar subsets of residues in proteins of known structure. However, the lack of functional and structural information on single residues, together with the low level of integration of this information in comparison methods, is a limitation that prevents these methods from being fully exploited in high-throughput analyses. Results Here we describe Query3d, a program that is both a structural DBMS (Database Management System) and a local comparison method. The method conserves a copy of all the residues of the Protein Data Bank annotated with a variety of functional and structural information. New annotations can be easily added from a variety of methods and known databases. The algorithm makes it possible to create complex queries based on the residues' function and then to compare only subsets of the selected residues. Functional information is also essential to speed up the comparison and the analysis of the results. Conclusion With Query3d, users can easily obtain statistics on how many and which residues share certain properties in all proteins of known structure. At the same time, the method also finds their structural neighbours in the whole PDB. Programs and data can be accessed through the PdbFun web interface. PMID:16351754

Prior knowledge based mining functional modules from Yeast PPI networks with gene ontology

PubMed Central

2010-01-01

Background In the literature, there are fruitful algorithmic approaches for identification functional modules in protein-protein interactions (PPI) networks. Because of accumulation of large-scale interaction data on multiple organisms and non-recording interaction data in the existing PPI database, it is still emergent to design novel computational techniques that can be able to correctly and scalably analyze interaction data sets. Indeed there are a number of large scale biological data sets providing indirect evidence for protein-protein interaction relationships. Results The main aim of this paper is to present a prior knowledge based mining strategy to identify functional modules from PPI networks with the aid of Gene Ontology. Higher similarity value in Gene Ontology means that two gene products are more functionally related to each other, so it is better to group such gene products into one functional module. We study (i) to encode the functional pairs into the existing PPI networks; and (ii) to use these functional pairs as pairwise constraints to supervise the existing functional module identification algorithms. Topology-based modularity metric and complex annotation in MIPs will be used to evaluate the identified functional modules by these two approaches. Conclusions The experimental results on Yeast PPI networks and GO have shown that the prior knowledge based learning methods perform better than the existing algorithms. PMID:21172053

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

PubMed Central

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

Comparison of molecular dynamics and superfamily spaces of protein domain deformation.

PubMed

Velázquez-Muriel, Javier A; Rueda, Manuel; Cuesta, Isabel; Pascual-Montano, Alberto; Orozco, Modesto; Carazo, José-María

2009-02-17

It is well known the strong relationship between protein structure and flexibility, on one hand, and biological protein function, on the other hand. Technically, protein flexibility exploration is an essential task in many applications, such as protein structure prediction and modeling. In this contribution we have compared two different approaches to explore the flexibility space of protein domains: i) molecular dynamics (MD-space), and ii) the study of the structural changes within superfamily (SF-space). Our analysis indicates that the MD-space and the SF-space display a significant overlap, but are still different enough to be considered as complementary. The SF-space space is wider but less complex than the MD-space, irrespective of the number of members in the superfamily. Also, the SF-space does not sample all possibilities offered by the MD-space, but often introduces very large changes along just a few deformation modes, whose number tend to a plateau as the number of related folds in the superfamily increases. Theoretically, we obtained two conclusions. First, that function restricts the access to some flexibility patterns to evolution, as we observe that when a superfamily member changes to become another, the path does not completely overlap with the physical deformability. Second, that conformational changes from variation in a superfamily are larger and much simpler than those allowed by physical deformability. Methodologically, the conclusion is that both spaces studied are complementary, and have different size and complexity. We expect this fact to have application in fields as 3D-EM/X-ray hybrid models or ab initio protein folding.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

PubMed

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

PubMed Central

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-01-01

Background Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. Results As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. Conclusion We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s). PMID:19055778

Protein disorder in the human diseasome: unfoldomics of human genetic diseases

PubMed Central

Midic, Uros; Oldfield, Christopher J; Dunker, A Keith; Obradovic, Zoran; Uversky, Vladimir N

2009-01-01

Background Intrinsically disordered proteins lack stable structure under physiological conditions, yet carry out many crucial biological functions, especially functions associated with regulation, recognition, signaling and control. Recently, human genetic diseases and related genes were organized into a bipartite graph (Goh KI, Cusick ME, Valle D, Childs B, Vidal M, et al. (2007) The human disease network. Proc Natl Acad Sci U S A 104: 8685–8690). This diseasome network revealed several significant features such as the common genetic origin of many diseases. Methods and findings We analyzed the abundance of intrinsic disorder in these diseasome network proteins by means of several prediction algorithms, and we analyzed the functional repertoires of these proteins based on prior studies relating disorder to function. Our analyses revealed that (i) Intrinsic disorder is common in proteins associated with many human genetic diseases; (ii) Different disease classes vary in the IDP contents of their associated proteins; (iii) Molecular recognition features, which are relatively short loosely structured protein regions within mostly disordered sequences and which gain structure upon binding to partners, are common in the diseasome, and their abundance correlates with the intrinsic disorder level; (iv) Some disease classes have a significant fraction of genes affected by alternative splicing, and the alternatively spliced regions in the corresponding proteins are predicted to be highly disordered; and (v) Correlations were found among the various diseasome graph-related properties and intrinsic disorder. Conclusion These observations provide the basis for the construction of the human-genetic-disease-associated unfoldome. PMID:19594871

The glassy state of crambin and the THz time scale protein-solvent fluctuations possibly related to protein function

PubMed Central

2014-01-01

Background THz experiments have been used to characterize the picosecond time scale fluctuations taking place in the model, globular protein crambin. Results Using both hydration and temperature as an experimental parameter, we have identified collective fluctuations (<= 200 cm−1) in the protein. Observation of the protein dynamics in the THz spectrum from both below and above the glass transition temperature (Tg) has provided unique insight into the microscopic interactions and modes that permit the solvent to effectively couple to the protein thermal fluctuations. Conclusions Our findings suggest that the solvent dynamics on the picosecond time scale not only contribute to protein flexibility but may also delineate the types of fluctuations that are able to form within the protein structure. PMID:25184036

A Score of the Ability of a Three-Dimensional Protein Model to Retrieve Its Own Sequence as a Quantitative Measure of Its Quality and Appropriateness

PubMed Central

Martínez-Castilla, León P.; Rodríguez-Sotres, Rogelio

2010-01-01

Background Despite the remarkable progress of bioinformatics, how the primary structure of a protein leads to a three-dimensional fold, and in turn determines its function remains an elusive question. Alignments of sequences with known function can be used to identify proteins with the same or similar function with high success. However, identification of function-related and structure-related amino acid positions is only possible after a detailed study of every protein. Folding pattern diversity seems to be much narrower than sequence diversity, and the amino acid sequences of natural proteins have evolved under a selective pressure comprising structural and functional requirements acting in parallel. Principal Findings The approach described in this work begins by generating a large number of amino acid sequences using ROSETTA [Dantas G et al. (2003) J Mol Biol 332:449–460], a program with notable robustness in the assignment of amino acids to a known three-dimensional structure. The resulting sequence-sets showed no conservation of amino acids at active sites, or protein-protein interfaces. Hidden Markov models built from the resulting sequence sets were used to search sequence databases. Surprisingly, the models retrieved from the database sequences belonged to proteins with the same or a very similar function. Given an appropriate cutoff, the rate of false positives was zero. According to our results, this protocol, here referred to as Rd.HMM, detects fine structural details on the folding patterns, that seem to be tightly linked to the fitness of a structural framework for a specific biological function. Conclusion Because the sequence of the native protein used to create the Rd.HMM model was always amongst the top hits, the procedure is a reliable tool to score, very accurately, the quality and appropriateness of computer-modeled 3D-structures, without the need for spectroscopy data. However, Rd.HMM is very sensitive to the conformational features of the models' backbone. PMID:20830209

Pattern similarity study of functional sites in protein sequences: lysozymes and cystatins

PubMed Central

Nakai, Shuryo; Li-Chan, Eunice CY; Dou, Jinglie

2005-01-01

Background Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. Results Hydrophobicity and β-turn propensity of reference segments with 3–7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. Conclusion Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available. PMID:15904486

An Ancestral Role for CONSTITUTIVE TRIPLE RESPONSE1 Proteins in Both Ethylene and Abscisic Acid Signaling1[OPEN

PubMed Central

Yasumura, Yuki; Pierik, Ronald; Kelly, Steven; Sakuta, Masaaki; Voesenek, Laurentius A.C.J.; Harberd, Nicholas P.

2015-01-01

Land plants have evolved adaptive regulatory mechanisms enabling the survival of environmental stresses associated with terrestrial life. Here, we focus on the evolution of the regulatory CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) component of the ethylene signaling pathway that modulates stress-related changes in plant growth and development. First, we compare CTR1-like proteins from a bryophyte, Physcomitrella patens (representative of early divergent land plants), with those of more recently diverged lycophyte and angiosperm species (including Arabidopsis [Arabidopsis thaliana]) and identify a monophyletic CTR1 family. The fully sequenced P. patens genome encodes only a single member of this family (PpCTR1L). Next, we compare the functions of PpCTR1L with that of related angiosperm proteins. We show that, like angiosperm CTR1 proteins (e.g. AtCTR1 of Arabidopsis), PpCTR1L modulates downstream ethylene signaling via direct interaction with ethylene receptors. These functions, therefore, likely predate the divergence of the bryophytes from the land-plant lineage. However, we also show that PpCTR1L unexpectedly has dual functions and additionally modulates abscisic acid (ABA) signaling. In contrast, while AtCTR1 lacks detectable ABA signaling functions, Arabidopsis has during evolution acquired another homolog that is functionally distinct from AtCTR1. In conclusion, the roles of CTR1-related proteins appear to have functionally diversified during land-plant evolution, and angiosperm CTR1-related proteins appear to have lost an ancestral ABA signaling function. Our study provides new insights into how molecular events such as gene duplication and functional differentiation may have contributed to the adaptive evolution of regulatory mechanisms in plants. PMID:26243614

Lack of Spartin Protein in Troyer Syndrome

PubMed Central

Bakowska, Joanna C.; Wang, Heng; Xin, Baozhong; Sumner, Charlotte J.; Blackstone, Craig

2017-01-01

Background Hereditary spastic paraplegias (SPG1-SPG33) are characterized by progressive spastic weakness of the lower limbs. A nucleotide deletion (1110delA) in the (SPG20; OMIM 275900) spartin gene is the origin of autosomal recessive Troyer syndrome. This mutation is predicted to cause premature termination of the spartin protein. However, it remains unknown whether this truncated spartin protein is absent or is present and partially functional in patients. Objective To determine whether the truncated spartin protein is present or absent in cells derived from patients with Troyer syndrome. Design Case report. Setting Academic research. Patients We describe a new family with Troyer syndrome due to the 1110delA mutation. Main Outcome Measures We cultured primary fibroblasts and generated lymphoblasts from affected individuals, carriers, and control subjects and subjected these cells to immunoblot analyses. Results Spartin protein is undetectable in several cell lines derived from patients with Troyer syndrome. Conclusions Our data suggest that Troyer syndrome results from complete loss of spartin protein rather than from the predicted partly functional fragment. This may reflect increased protein degradation or impaired translation. PMID:18413476

Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leite, Wellington C.; Galvão, Carolina W.; Saab, Sérgio C.

The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminalmore » polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.« less

Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

PubMed Central

2015-01-01

Background Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. Results One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. Conclusions These results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and allow a better understanding of the emergence of biological diversity. PMID:26693737

The pilus usher controls protein interactions via domain masking and is functional as an oligomer.

PubMed

Werneburg, Glenn T; Henderson, Nadine S; Portnoy, Erica B; Sarowar, Samema; Hultgren, Scott J; Li, Huilin; Thanassi, David G

2015-07-01

The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

The Pilus Usher Controls Protein Interactions via Domain Masking and is Functional as an Oligomer

PubMed Central

Werneburg, Glenn T.; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Li, Huilin; Thanassi, David G.

2015-01-01

The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria. PMID:26052892

CENPI is overexpressed in colorectal cancer and regulates cell migration and invasion.

PubMed

Ding, Na; Li, Rongxin; Shi, Wenhao; He, Cui

2018-06-21

Centromere protein I (CENPI),an important member of centromere protein family, has been suggest to serve as a oncogene in breast cancer, but the clinical significance and biological function of CENPI in colorectal cancer (CRC) is still unclear. In our results, we found CENPI was overexpressed in CRC tissues and cells, and associated with clinical stage, tumor depth, lymph node metastasis, distant metastasis and differentiation in CRC patients. However, there was no significant association between CENPI protein expression and overall survival time in colon cancer patients and rectal cancer patients through analyzing TCGA survival data. Moreover, CENPI mRNA and protein were increased in metastatic lymph nodes compared with primary CRC tissues. Down-regulation of CENPI expression suppresses CRC cell migration, invasion and epithelial mesenchymal transition process. In conclusion, CENPI is overexpressed in CRC and functions as oncogene in modulating CRC cell migration, invasion and EMT process. Copyright © 2018. Published by Elsevier B.V.

Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes

PubMed Central

Bridges, Dave; Fraser, Marie E; Moorhead, Greg BG

2005-01-01

Background Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules. Results Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals. Conclusion This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants. PMID:15644130

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

PubMed Central

Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

2009-01-01

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

Biochemical and Functional Analysis of Drosophila-Sciara Chimeric Sex-Lethal Proteins

PubMed Central

Ruiz, María Fernanda; Sarno, Francesca; Zorrilla, Silvia; Rivas, Germán; Sánchez, Lucas

2013-01-01

Background The Drosophila SXL protein controls sex determination and dosage compensation. It is a sex-specific factor controlling splicing of its own Sxl pre-mRNA (auto-regulation), tra pre-mRNA (sex determination) and msl-2 pre-mRNA plus translation of msl-2 mRNA (dosage compensation). Outside the drosophilids, the same SXL protein has been found in both sexes so that, in the non-drosophilids, SXL does not appear to play the key discriminating role in sex determination and dosage compensation that it plays in Drosophila. Comparison of SXL proteins revealed that its spatial organisation is conserved, with the RNA-binding domains being highly conserved, whereas the N- and C-terminal domains showing significant variation. This manuscript focuses on the evolution of the SXL protein itself and not on regulation of its expression. Methodology Drosophila-Sciara chimeric SXL proteins were produced. Sciara SXL represents the non-sex-specific function of ancient SXL in the non-drosophilids from which presumably Drosophila SXL evolved. Two questions were addressed. Did the Drosophila SXL protein have affected their functions when their N- and C-terminal domains were replaced by the corresponding ones of Sciara? Did the Sciara SXL protein acquire Drosophila sex-specific functions when the Drosophila N- and C-terminal domains replaced those of Sciara? The chimeric SXL proteins were analysed in vitro to study their binding affinity and cooperative properties, and in vivo to analyse their effect on sex determination and dosage compensation by producing Drosophila flies that were transgenic for the chimeric SXL proteins. Conclusions The sex-specific properties of extant Drosophila SXL protein depend on its global structure rather than on a specific domain. This implies that the modifications, mainly in the N- and C-terminal domains, that occurred in the SXL protein during its evolution within the drosophilid lineage represent co-evolutionary changes that determine the appropriate folding of SXL to carry out its sex-specific functions. PMID:23762307

The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function.

PubMed

Thundathil, J C; Rajamanickam, G D; Kastelic, J P; Newton, L D

2012-08-01

Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility. Bovine testes must be maintained 4-5 °C below body-core temperature for normal spermatogenesis. The effects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identified a cohort of sperm functional proteins differentially expressed between normal vs abnormal sperm, including a testis-specific isoform of Na(+) /K(+) -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na(+) /K(+) -ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function. © 2012 Blackwell Verlag GmbH.

Short-term in vivo inhibition of nitric oxide synthase with L-NAME influences the contractile function of single left ventricular myocytes in rats.

PubMed

Lunz, Wellington; Natali, Antônio José; Carneiro, Miguel Araújo; Dos Santos Aggum Capettini, Luciano; Baldo, Marcelo Perim; de Souza, Matheus Ornelas; Quintão, Judson Fonseca; Bozi, Luiz Henrique Marchesi; Lemos, Virginia Soares; Mill, José Geraldo

2011-04-01

The main purpose of this study was to investigate the effects of short-term L-NAME treatment on the contractile function of left ventricle (LV) myocytes and the expression of proteins related to Ca(2+) homeostasis. Data from Wistar rats treated with L-NAME (L group, n = 20; 0.7 g/L in drinking water; 7 days) were compared with results from untreated controls (C group, n = 20). Cardiomyocytes from the L group showed increased (p < 0.05) fractional shortening (23%) and maximum rate of shortening (20%) compared with the C group. LV from the L group also showed increased (p < 0.05) expression of the ryanodine receptor 2 and Na(+)/Ca(2+) exchanger proteins (76% and 83%, respectively; p < 0.05). However, the L and C groups showed similar in vivo hemodynamic parameters of cardiac function. In conclusion, short-term NOS inhibition determines an increased expression of Ca(2+) regulatory proteins, which contributes to improving cardiomyocyte contractile function, preserving left ventricular function.

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

PubMed Central

Vincent, Grace; Lamon, Séverine; Gant, Nicholas; Vincent, Peter J.; MacDonald, Julia R.; Markworth, James F.; Edge, Johann A.; Hickey, Anthony J. R.

2015-01-01

Purpose: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. Methods: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. Results: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. Conclusion: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression. PMID:25759671

Identification of functional interactome of a key cell division regulatory protein CedA of E.coli.

PubMed

Sharma, Pankaj; Tomar, Anil Kumar; Kundu, Bishwajit

2018-01-01

Cell division is compromised in DnaAcos mutant Escherichia coli cells that results in filamentous cell morphology. This is countered by over-expression of CedA protein that induces cytokinesis and thus, regular cell morphology is regained; however via an unknown mechanism. To understand the process systematically, exact role of CedA should be deciphered. Protein interactions are crucial for functional organization of a cell and their identification helps in revealing exact function(s) of a protein and its binding partners. Thus, this study was intended to identify CedA binding proteins (CBPs) to gain more clues of CedA function. We isolated CBPs by pull down assay using purified recombinant CedA and identified nine CBPs by mass spectrometric analysis (MALDI-TOF MS and LC-MS/MS), viz. PDHA1, RL2, DNAK, LPP, RPOB, G6PD, GLMS, RL3 and YBCJ. Based on CBPs identified, we hypothesize that CedA plays a crucial and multifaceted role in cell cycle regulation and specific pathways in which CedA participates may include transcription and energy metabolism. However, further validation through in-vitro and in-vivo experiments is necessary. In conclusion, identification of CBPs may help us in deciphering mechanism of CedA mediated cell division during chromosomal DNA over-replication. Copyright © 2017 Elsevier B.V. All rights reserved.

Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

PubMed Central

Aparicio, I. M.; Espino, J.; Bejarano, I.; Gallardo-Soler, A.; Campo, M. L.; Salido, G. M.; Pariente, J. A.; Peña, F. J.; Tapia, J. A.

2016-01-01

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis. PMID:27633131

Calcineurin Regulates Myocardial Function during Acute Endotoxemia

PubMed Central

Joshi, Mandar S.; Julian, Mark W.; Huff, Jennifer E.; Bauer, John A.; Xia, Yong; Crouser, Elliott D.

2006-01-01

Rationale: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT). Objectives: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function. Methods: Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function. Measurements and Main Results: LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia. Conclusions: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia. PMID:16424445

The relative vertex clustering value - a new criterion for the fast discovery of functional modules in protein interaction networks

PubMed Central

2015-01-01

Background Cellular processes are known to be modular and are realized by groups of proteins implicated in common biological functions. Such groups of proteins are called functional modules, and many community detection methods have been devised for their discovery from protein interaction networks (PINs) data. In current agglomerative clustering approaches, vertices with just a very few neighbors are often classified as separate clusters, which does not make sense biologically. Also, a major limitation of agglomerative techniques is that their computational efficiency do not scale well to large PINs. Finally, PIN data obtained from large scale experiments generally contain many false positives, and this makes it hard for agglomerative clustering methods to find the correct clusters, since they are known to be sensitive to noisy data. Results We propose a local similarity premetric, the relative vertex clustering value, as a new criterion allowing to decide when a node can be added to a given node's cluster and which addresses the above three issues. Based on this criterion, we introduce a novel and very fast agglomerative clustering technique, FAC-PIN, for discovering functional modules and protein complexes from a PIN data. Conclusions Our proposed FAC-PIN algorithm is applied to nine PIN data from eight different species including the yeast PIN, and the identified functional modules are validated using Gene Ontology (GO) annotations from DAVID Bioinformatics Resources. Identified protein complexes are also validated using experimentally verified complexes. Computational results show that FAC-PIN can discover functional modules or protein complexes from PINs more accurately and more efficiently than HC-PIN and CNM, the current state-of-the-art approaches for clustering PINs in an agglomerative manner. PMID:25734691

HMPAS: Human Membrane Protein Analysis System

PubMed Central

2013-01-01

Background Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups. Methods We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences. Results We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins. Conclusions HMPAS provides comprehensive information about human membrane proteins including specific features of certain membrane protein groups. In this system, user can acquire the information of individual proteins and specified groups focused on their conserved sequence features, involved cellular processes, and diseases. HMPAS may contribute as a valuable resource for the inference of novel cellular mechanisms and pharmaceutical targets associated with the human membrane proteins. HMPAS is freely available at http://fcode.kaist.ac.kr/hmpas. PMID:24564858

[Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter].

PubMed

Song, Jian; Liu, Xueming; Li, Jiada; Liu, Huadie; Peng, Zhen; Chen, Hongsheng; Mei, Lingyun; He, Chufeng; Feng, Yong

2018-05-28

To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR) promoter.
 Methods: The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, respectively.
 Results: IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted.
 Conclusion: IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.

Salt-Induced Universal Slowing Down of the Short-Time Self-Diffusion of a Globular Protein in Aqueous Solution

DOE PAGES

Grimaldo, Marco; Roosen-Runge, Felix; Hennig, Marcus; ...

2015-06-17

The short-time self-diffusion D of the globular model protein bovine serum albumin in aqueous (D 2O) solutions has been measured comprehensively as a function of the protein and trivalent salt (YCl 3) concentration, noted c p and c s, respectively. We observe that D follows a universal master curve D(c s,c p) = D(c s = 0,c p) g(c s/c p), where D(c s= 0,c p) is the diffusion coefficient in the absence of salt and g(c s/c p) is a scalar function solely depending on the ratio of the salt and protein concentration. This observation is consistent with amore » universal scaling of the bonding probability in a picture of cluster formation of patchy particles. In conclusion, the finding corroborates the predictive power of the description of proteins as colloids with distinct attractive ion-activated surface patches.« less

An Improved, Bias-Reduced Probabilistic Functional Gene Network of Baker's Yeast, Saccharomyces cerevisiae

PubMed Central

Lee, Insuk; Li, Zhihua; Marcotte, Edward M.

2007-01-01

Background Probabilistic functional gene networks are powerful theoretical frameworks for integrating heterogeneous functional genomics and proteomics data into objective models of cellular systems. Such networks provide syntheses of millions of discrete experimental observations, spanning DNA microarray experiments, physical protein interactions, genetic interactions, and comparative genomics; the resulting networks can then be easily applied to generate testable hypotheses regarding specific gene functions and associations. Methodology/Principal Findings We report a significantly improved version (v. 2) of a probabilistic functional gene network [1] of the baker's yeast, Saccharomyces cerevisiae. We describe our optimization methods and illustrate their effects in three major areas: the reduction of functional bias in network training reference sets, the application of a probabilistic model for calculating confidences in pair-wise protein physical or genetic interactions, and the introduction of simple thresholds that eliminate many false positive mRNA co-expression relationships. Using the network, we predict and experimentally verify the function of the yeast RNA binding protein Puf6 in 60S ribosomal subunit biogenesis. Conclusions/Significance YeastNet v. 2, constructed using these optimizations together with additional data, shows significant reduction in bias and improvements in precision and recall, in total covering 102,803 linkages among 5,483 yeast proteins (95% of the validated proteome). YeastNet is available from http://www.yeastnet.org. PMID:17912365

Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling

PubMed Central

Zhou, Jian-Ying; Krovvidi, Ravi K.; Gao, Yuqian; Gao, Hong; Petritis, Brianne O.; De, Asit; Miller-Graziano, Carol; Bankey, Paul E.; Petyuk, Vladislav A.; Nicora, Carrie D.; Clauss, Therese R; Moore, Ronald J.; Shi, Tujin; Brown, Joseph N.; Kaushal, Amit; Xiao, Wenzhong; Davis, Ronald W.; Maier, Ronald V.; Tompkins, Ronald G.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.

2013-01-01

PURPOSE Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. EXPERIMENTAL DESIGN We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. RESULTS A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. CONCLUSIONS AND CLINICAL RELEVANCE Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs. PMID:23589343

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

DOE PAGES

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos; ...

2015-10-26

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

N-ethylmaleimide-sensitive factor interacts with the serotonin transporter and modulates its trafficking: implications for pathophysiology in autism

PubMed Central

2014-01-01

Background Changes in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism. Methods Novel SERT-binding proteins were examined by a pull-down system. Alterations of SERT function and membrane expression upon knockdown of the novel SERT-binding protein were studied in HEK293-hSERT cells. Endogenous interaction of SERT with the protein was evaluated in mouse brains. Alterations in the mRNA expression of SERT (SLC6A4) and the SERT-binding protein in the post-mortem brains and the lymphocytes of autism patients were compared to nonclinical controls. Results N-ethylmaleimide-sensitive factor (NSF) was identified as a novel SERT-binding protein. NSF was co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and decreased SERT uptake function. NSF was endogenously co-localized with SERT and interacted with SERT. While SLC6A4 expression was not significantly changed, NSF expression tended to be reduced in post-mortem brains, and was significantly reduced in lymphocytes of autistic subjects, which correlated with the severity of the clinical symptoms. Conclusions These data clearly show that NSF interacts with SERT under physiological conditions and is required for SERT membrane trafficking and uptake function. A possible role for NSF in the pathophysiology of autism through modulation of SERT trafficking, is suggested. PMID:24834316

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giuliani, Sarah E; Frank, Ashley M; Corgliano, Danielle M

Abstract Background: Transporter proteins are one of an organism s primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks. The functional annotation of most transporters is currently limited to general classification into families. The development of capabilities to map ligands with specific transporters would improve our knowledge of the function of these proteins, improve the annotation of related genomes, and facilitate predictions for their role in cellular responses to environmental changes. Results: To improve the utility of the functional annotation for ABC transporters, we expressed and purifiedmore » the set of solute binding proteins from Rhodopseudomonas palustris and characterized their ligand-binding specificity. Our approach utilized ligand libraries consisting of environmental and cellular metabolic compounds, and fluorescence thermal shift based high throughput ligand binding screens. This process resulted in the identification of specific binding ligands for approximately 64% of the purified and screened proteins. The collection of binding ligands is representative of common functionalities associated with many bacterial organisms as well as specific capabilities linked to the ecological niche occupied by R. palustris. Conclusion: The functional screen identified specific ligands that bound to ABC transporter periplasmic binding subunits from R. palustris. These assignments provide unique insight for the metabolic capabilities of this organism and are consistent with the ecological niche of strain isolation. This functional insight can be used to improve the annotation of related organisms and provides a route to evaluate the evolution of this important and diverse group of transporter proteins.« less

Interfacing cellular networks of S. cerevisiae and E. coli: Connecting dynamic and genetic information

PubMed Central

2013-01-01

Background In recent years, various types of cellular networks have penetrated biology and are nowadays used omnipresently for studying eukaryote and prokaryote organisms. Still, the relation and the biological overlap among phenomenological and inferential gene networks, e.g., between the protein interaction network and the gene regulatory network inferred from large-scale transcriptomic data, is largely unexplored. Results We provide in this study an in-depth analysis of the structural, functional and chromosomal relationship between a protein-protein network, a transcriptional regulatory network and an inferred gene regulatory network, for S. cerevisiae and E. coli. Further, we study global and local aspects of these networks and their biological information overlap by comparing, e.g., the functional co-occurrence of Gene Ontology terms by exploiting the available interaction structure among the genes. Conclusions Although the individual networks represent different levels of cellular interactions with global structural and functional dissimilarities, we observe crucial functions of their network interfaces for the assembly of protein complexes, proteolysis, transcription, translation, metabolic and regulatory interactions. Overall, our results shed light on the integrability of these networks and their interfacing biological processes. PMID:23663484

Geraniol promotes functional recovery and attenuates neuropathic pain in rats with spinal cord injury.

PubMed

Lv, Yan; Zhang, Liang; Li, Na; Mai, Naiken; Zhang, Yu; Pan, Shuyi

2017-12-01

Geraniol, a plant-derived monoterpene, has been extensively studied and showed a wide variety of beneficial effects. The aim of this study was to investigate the therapeutic effect of geraniol on functional recovery and neuropathic pain in rats with spinal cord injury (SCI). Rats received a clip-compression SCI and were treated with geraniol 6 h following SCI. Treatment of SCI rats with geraniol markedly improved locomotor function, and reduced sensitivity to the mechanical allodynia and thermal hyperalgesia. Treatment of SCI rats with geraniol increased NeuN-positive cells, suppressed expression of glial fibrillary acidic protein, and reduced activity of caspase-3 in the injured region. Treatment of SCI rats with geraniol reduced levels of malondialdehyde and 3-nitrotyrosine, upregulated protein expression of nuclear factor-erythroid 2-related factor 2 and heme oxygenase 1, and suppressed expression of inducible nitric oxide synthase in the injured region. In addition, treatment of SCI rats with geraniol downregulated protein expression of N-methyl-d-aspartate receptor 1 and reduced the number of CD68-positive cells and protein levels of TNF-α in the injured region. In conclusion, geraniol significantly promoted the recovery of neuronal function and attenuated neuropathic pain after SCI.

Molecular and ultrastructural analysis of forisome subunits reveals the principles of forisome assembly

PubMed Central

Müller, Boje; Groscurth, Sira; Menzel, Matthias; Rüping, Boris A.; Twyman, Richard M.; Prüfer, Dirk; Noll, Gundula A.

2014-01-01

Background and Aims Forisomes are specialized structural phloem proteins that mediate sieve element occlusion after wounding exclusively in papilionoid legumes, but most studies of forisome structure and function have focused on the Old World clade rather than the early lineages. A comprehensive phylogenetic, molecular, structural and functional analysis of forisomes from species covering a broad spectrum of the papilionoid legumes was therefore carried out, including the first analysis of Dipteryx panamensis forisomes, representing the earliest branch of the Papilionoideae lineage. The aim was to study the molecular, structural and functional conservation among forisomes from different tribes and to establish the roles of individual forisome subunits. Methods Sequence analysis and bioinformatics were combined with structural and functional analysis of native forisomes and artificial forisome-like protein bodies, the latter produced by expressing forisome genes from different legumes in a heterologous background. The structure of these bodies was analysed using a combination of confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the function of individual subunits was examined by combinatorial expression, micromanipulation and light microscopy. Key Results Dipteryx panamensis native forisomes and homomeric protein bodies assembled from the single sieve element occlusion by forisome (SEO-F) subunit identified in this species were structurally and functionally similar to forisomes from the Old World clade. In contrast, homomeric protein bodies assembled from individual SEO-F subunits from Old World species yielded artificial forisomes differing in proportion to their native counterparts, suggesting that multiple SEO-F proteins are required for forisome assembly in these plants. Structural differences between Medicago truncatula native forisomes, homomeric protein bodies and heteromeric bodies containing all possible subunit combinations suggested that combinations of SEO-F proteins may fine-tune the geometric proportions and reactivity of forisomes. Conclusions It is concluded that forisome structure and function have been strongly conserved during evolution and that species-dependent subsets of SEO-F proteins may have evolved to fine-tune the structure of native forisomes. PMID:24694827

Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

PubMed Central

Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

2012-01-01

It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

Small Proteins Can No Longer Be Ignored

PubMed Central

Storz, Gisela; Wolf, Yuri I.; Ramamurthi, Kumaran S.

2014-01-01

Small proteins, here defined as proteins of 50 amino acids or less in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years however, increasing numbers of small proteins have been identified either through the realization that mutations in “intergenic” regions actually are within unannotated small protein genes, or through the discovery that some small, regulatory RNAs (sRNAs) encode small proteins. These insights together with comparative sequence analysis indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review will summarize what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which the small proteins act and pointing to several general conclusions. Important questions for future studies of these overlooked proteins also will be discussed. PMID:24606146

Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum.

PubMed

Xie, Longxiang; Fang, Wenjie; Deng, Wanyan; Yu, Zhaoxiao; Li, Juan; Chen, Min; Liao, Wanqing; Xie, Jianping; Pan, Weihua

2016-04-01

Histoplasma capsulatum is the causative agent of human histoplasmosis, which can cause respiratory and systemic mycosis in immune-compromised individuals. Lysine acetylation, a protein posttranslational protein modification, is widespread in both eukaryotes and prokaryotes. Although increasing evidence suggests that lysine acetylation may play critical roles in fungus physiology, very little is known about its extent and function in H. capsulatum. To comprehensively profile protein lysine acetylation in H. capsulatum, we performed a global acetylome analysis through peptide prefractionation, antibody enrichment, and LC-MS/MS analysis, identifying 775 acetylation sites on 456 acetylated proteins; and functionally analysis showing their involvement in different biological processes. We defined six types of acetylation site motifs, and the results imply that lysine residue of polypeptide with tyrosine at the -1 and +1 positions, histidine at the +1 position, and phenylalanine (F) at the +1 and +2 position is a preferred substrate of lysine acetyltransferase. Moreover, some virulence factors candidates including calmodulin and DnaK are acetylated. In conclusion, our data set may serve as an important resource for the elucidation of associations between functional protein lysine acetylation and virulence in H. capsulatum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Water dynamics in protein hydration shells: the molecular origins of the dynamical perturbation.

PubMed

Fogarty, Aoife C; Laage, Damien

2014-07-17

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra.

Water Dynamics in Protein Hydration Shells: The Molecular Origins of the Dynamical Perturbation

PubMed Central

2014-01-01

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra. PMID:24479585

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bienz, K.; Egger, D.; Troxler, M.

1990-03-01

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but didmore » not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.« less

Evaluation of erythroblast macrophage protein related to erythroblastic islands in patients with hematopoietic stem cell transplantation

PubMed Central

2013-01-01

Background Hematopoietic evaluation of the patients after Hematopoietic stem cell transplantation (HSCT) is very important. Erythroblast macrophage protein (Emp) is a key protein with function in normal differentiation of erythroid cells and macrophages. Emp expression correlates with erythroblastic island formation, a process widely believed to be associated with hematopoiesis in bone marrow. We aimed to investigate the hematopoietic function of bone marrow from 46 HSCT patients and 16 inpatients with severe anemia applied to the treatment of EPO by measuring Emp expression level. Methods Emp mRNA and protein expression levels in mononuclear cells of bone marrow and peripheral blood samples were detected by RT-PCR and Western blotting method respectively. Results While hematopoiesis occurs in bone marrow, Emp expression level was elevated and more erythroblastic islands were found , and Emp is upregulated in bone marrow in response to erythropoietin (EPO) treatment. Conclusions Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Emp could be a potential biomarker for hematopoietic evaluation of HSCT patients. PMID:23566571

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

PubMed

Besbrugge, Nienke; Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; Kulkarni, Shubhada R; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Bontinck, Michiel; Aesaert, Stijn; Impens, Francis; Gevaert, Kris; Van Damme, Daniel; Van Lijsebettens, Mieke; Inzé, Dirk; Vandepoele, Klaas; Nelissen, Hilde; De Jaeger, Geert

2018-06-01

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research. © 2018 American Society of Plant Biologists. All rights reserved.

In vitro digestibility, protein composition and techno-functional properties of Saskatchewan grown yellow field peas (Pisum sativum L.) as affected by processing.

PubMed

Ma, Zhen; Boye, Joyce I; Hu, Xinzhong

2017-02-01

Saskatchewan grown yellow field pea was subjected to different processing conditions including dehulling, micronization, roasting, conventional/microwave cooking, germination, and combined germination and conventional cooking/roasting. Their nutritional and antinutritional compositions, functional properties, microstructure, thermal properties, in vitro protein and starch digestibility, and protein composition were studied. Processed field peas including conventional cooked yellow peas (CCYP), microwave cooked yellow peas (MCYP), germinated-conventional cooked yellow peas (GCCYP), and germinated-roasted yellow peas (GRYP) exhibited the significantly higher in vitro protein digestibility (IVPD), which was in accordance with their significantly lower trypsin inhibitor activity and tannin content. The SDS-PAGE and size exclusion HPLC profiles of untreated pea proteins and their hydrolysates also confirmed the IVPD result that these four treatments facilitated the hydrolysis of pea proteins to a greater extent. The CCYP, MCYP, GCCYP, and GRYP also exhibited significantly higher starch digestibility which was supported by their lower onset (T o ), peak (T p ), and conclusion (T c ) temperatures obtained from DSC thermogram, their lower pasting properties and starch damage results, as well as their distinguished amorphous flakes' configuration observed on the scanning electron microscopic image. LC/ESI-MS/MS analysis following in-gel digests of SDS-PAGE separated proteins allowed detailed compositional characterization of pea proteins. The present study would provide fundamental information to help to better understand the functionality of field peas as ingredients, and particularly in regards to agri-food industry to improve the process efficiency of field peas with enhanced nutritional and techno-functional qualities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative proteomic analysis of outer membrane protein 43 (omp43)-deficient Bartonella henselae.

PubMed

Kang, Jun-Gu; Lee, Hee-Woo; Ko, Sungjin; Chae, Joon-Seok

2018-01-31

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae -derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δ omp 43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae . To study the differences in proteomic expression between WT and Δ omp 43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tld D, efp, ntr X, pdh A, pur B, and ATPA mRNA expression and decreases in Rho and yfe A mRNA expression were confirmed in Δ omp 43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.

An efficient algorithm for pairwise local alignment of protein interaction networks

DOE PAGES

Chen, Wenbin; Schmidt, Matthew; Tian, Wenhong; ...

2015-04-01

Recently, researchers seeking to understand, modify, and create beneficial traits in organisms have looked for evolutionarily conserved patterns of protein interactions. Their conservation likely means that the proteins of these conserved functional modules are important to the trait's expression. In this paper, we formulate the problem of identifying these conserved patterns as a graph optimization problem, and develop a fast heuristic algorithm for this problem. We compare the performance of our network alignment algorithm to that of the MaWISh algorithm [Koyuturk M, Kim Y, Topkara U, Subramaniam S, Szpankowski W, Grama A, Pairwise alignment of protein interaction networks, J Computmore » Biol 13(2): 182-199, 2006.], which bases its search algorithm on a related decision problem formulation. We find that our algorithm discovers conserved modules with a larger number of proteins in an order of magnitude less time. In conclusion, the protein sets found by our algorithm correspond to known conserved functional modules at comparable precision and recall rates as those produced by the MaWISh algorithm.« less

Relation of Response to Treatment with Dorzolamide in X-linked Retinoschisis to the Mechanism of Functional Loss in Retinoschisin

PubMed Central

Walia, Saloni; Fishman, Gerald A.; Molday, Robert S.; Dyka, Frank M.; Kumar, Nalin M.; Ehlinger, Mary A.; Stone, Edwin M.

2009-01-01

PURPOSE To determine if a positive response of macular cysts to treatment with dorzolamide eye drops in patients with juvenile X-linked retinoschisis (XLRS) can occur with mutations which result in different types of retinoschisin protein dysfunction. DESIGN Retrospective case series METHODS Thirteen eyes of 7 patients seen at the University of Illinois at Chicago with a known diagnosis of XLRS were included. Each patient had prior or was on current treatment with topical dorzolamide. One patient from each family was screened for a genetic mutation. Using the method of cell transfection and protein preparation, the mutation in each patient was further analyzed and categorized into three groups: 1. total absence of retinoschisin protein secretion, 2. decreased expression of the secreted protein or 3. secretion of a non-functional protein. The response to dorzolamide was observed using optical coherence tomography. RESULTS Significant improvement in the foveal zone thickness was observed with the use of dorzolamide in 3 of 4 patients with absence of protein secretion, in 2 patients with a lack of protein expression and in 1 patient with a non-functional protein secretion. CONCLUSION This study shows that the response of macular cysts to dorzolamide in patients with XLRS may be observed independent of the mechanism responsible for retinoschisin protein dysfunction. Hence, treatment with dorzolamide may be effective in patients with different mechanisms of dysfunction in retinoschisin. PMID:18834580

In the Thick of It: HCM-Causing Mutations in Myosin Binding Proteins of the Thick Filament

PubMed Central

Harris, Samantha P.; Lyons, Ross G.; Bezold, Kristina L.

2010-01-01

In the 20 yrs since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM) an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins including the myosin essential and regulatory light chains and cardiac myosin binding protein-C (cMyBP-C). However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes. PMID:21415409

Phosphorylation of RACK1 in plants

DOE PAGES

Chen, Jay -Gui

2015-08-31

Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory systemmore » in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.« less

Development of a novel set of Gateway-compatible vectors for live imaging in insect cells.

PubMed

Maroniche, G A; Mongelli, V C; Alfonso, V; Llauger, G; Taboga, O; del Vas, Mariana

2011-10-01

Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

A S-Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self-Assembly.

PubMed

Wang, Xu-Ying; Wang, Dian-Bing; Zhang, Zhi-Ping; Bi, Li-Jun; Zhang, Ji-Bin; Ding, Wei; Zhang, Xian-En

2015-11-18

S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplification. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma Amyloid β-Protein and C-reactive Protein in Relation to the Rate of Progression of Alzheimer Disease

PubMed Central

Locascio, Joseph J.; Fukumoto, Hiroaki; Yap, Liang; Bottiglieri, Teodoro; Growdon, John H.; Hyman, Bradley T.; Irizarry, Michael C.

2008-01-01

Objective To examine whether plasma markers of amyloid precursor protein metabolism (amyloid β-protein ending in Val-40 [Aβ40] and Ala-42 [Aβ42]), inflammation (high-sensitivity C-reactive protein), and folic acid metabolism (folic acid, vitamin B12, and total homocysteine levels) are associated with the rate of cognitive and functional decline in persons with Alzheimer disease. Design Longitudinal study across a mean (SD) of 4.2 (2.6) years with assessments at approximately 6- to 12- month intervals. Setting Out patient care. Patients A cohort of 122 patients having a clinical diagnosis of probable Alzheimer disease, each with at least 2 assessments across time. Main Outcome Measures Scores on the cognitive Information-Memory-Concentration subscale of the Blessed Dementia Scale and the functional Weintraub Activities of Daily Living Scale. Results Low plasma levels of Aβ40, Aβ42, and high-sensitivity C-reactive protein were associated with a significantly more rapid cognitive decline, as indexed using the Blessed Dementia Scale, than were high levels. Low levels of Aβ42 and high-sensitivity C-reactive protein were significantly associated with more rapid functional decline on the Weintraub Activities of Daily Living Scale than were high levels. These plasma markers contributed about 5% to 12% of the variance accounted for on the Blessed Dementia Scale and the Activities of Daily Living Scale by fixed-effects predictors. Measures of folic acid metabolism were not associated with changes on either the Blessed Dementia Scale or the Activities of Daily Living Scale. Conclusions Plasma markers of amyloid precursor protein metabolism and C-reactive protein may be associated with the rate of cognitive and functional decline in patients with Alzheimer disease. PMID:18541797

FEZ2 Has Acquired Additional Protein Interaction Partners Relative to FEZ1: Functional and Evolutionary Implications

PubMed Central

Alborghetti, Marcos R.; Furlan, Ariane S.; Kobarg, Jörg

2011-01-01

Background The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function. Methodology/Principal Findings Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom. Conclusions/Significance We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins. PMID:21408165

Accounting for epistatic interactions improves the functional analysis of protein structures

PubMed Central

Wilkins, Angela D.; Venner, Eric; Marciano, David C.; Erdin, Serkan; Atri, Benu; Lua, Rhonald C.; Lichtarge, Olivier

2013-01-01

Motivation: The constraints under which sequence, structure and function coevolve are not fully understood. Bringing this mutual relationship to light can reveal the molecular basis of binding, catalysis and allostery, thereby identifying function and rationally guiding protein redesign. Underlying these relationships are the epistatic interactions that occur when the consequences of a mutation to a protein are determined by the genetic background in which it occurs. Based on prior data, we hypothesize that epistatic forces operate most strongly between residues nearby in the structure, resulting in smooth evolutionary importance across the structure. Methods and Results: We find that when residue scores of evolutionary importance are distributed smoothly between nearby residues, functional site prediction accuracy improves. Accordingly, we designed a novel measure of evolutionary importance that focuses on the interaction between pairs of structurally neighboring residues. This measure that we term pair-interaction Evolutionary Trace yields greater functional site overlap and better structure-based proteome-wide functional predictions. Conclusions: Our data show that the structural smoothness of evolutionary importance is a fundamental feature of the coevolution of sequence, structure and function. Mutations operate on individual residues, but selective pressure depends in part on the extent to which a mutation perturbs interactions with neighboring residues. In practice, this principle led us to redefine the importance of a residue in terms of the importance of its epistatic interactions with neighbors, yielding better annotation of functional residues, motivating experimental validation of a novel functional site in LexA and refining protein function prediction. Contact: lichtarge@bcm.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24021383

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

High-resolution structure of the Escherichia coli ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

High-resolution structure of the Escherichia coli ribosome

DOE PAGES

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

2015-03-16

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

PubMed Central

Malina, Halina Z; Hess, Otto M

2004-01-01

Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions. PMID:15068490

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

PubMed Central

2012-01-01

Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834

Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

PubMed

Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

2016-06-01

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. © 2016 The Authors Cellular Microbiology published by John Wiley & Sons Ltd.

Klotho protein lowered in senile patients with brady sinus arrhythmia

PubMed Central

Wang, Ying; Yang, Wei; Zheng, Ernv; Zhang, Wei; Su, Xianming

2015-01-01

Objective: To explore the correlationship between brady sinus arrhythmia and the levels of serum klotho protein in aged. Methods: 104 patients over 75 years old with brady sinus arrhythmia (experiment group) were enrolled, including 34 cases of sinus arrest, 43 cases of sinus bradycardia and 25 cases of atrioventricular block. 109 patients over 75 years old without brady sinus arrhymia were chosen as control group. All subjects were monitored by Holter. The levels of serum klotho protein were detected and compared among three groups. The correlation between the frequency of sinus arrest and the levels of serum klotho protein was analyzed simultaneously. Results: The levels of serum klotho protein in experiment group were lower than that in control group (P<0.01); the sinus arrest frequency was negatively correlated with the levels of serum klotho protien. The levels of serum klotho protein in patients with sinus arrest were lower than that with sinus bradycardia and atrioventricularblock (P<0.05). But there was no significant difference between sinus bradycardia group and atrioventricular block group. Conclusion: The levels of serum klotho protein may reflect the function of sinoatrial node and could be used as an index to estimate the function of sinoatrial node. PMID:26550342

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck].

PubMed

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-11-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

Exocyst Complex Protein Expression in the Human Placenta

PubMed Central

Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

2014-01-01

Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

The Role of the Progressive Ankylosis Protein (ANK) in Adipogenic/Osteogenic Fate Decision of Precursor Cells

PubMed Central

Minashima, Takeshi; Quirno, Martin; Lee, You Jin; Kirsch, Thorsten

2017-01-01

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation. PMID:28286238

GFP Loss-of-Function Mutations in Arabidopsis thaliana.

PubMed

Fu, Jason L; Kanno, Tatsuo; Liang, Shih-Chieh; Matzke, Antonius J M; Matzke, Marjori

2015-07-06

Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate-induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability. Copyright © 2015 Fu et al.

GFP Loss-of-Function Mutations in Arabidopsis thaliana

PubMed Central

Fu, Jason L.; Kanno, Tatsuo; Liang, Shih-Chieh; Matzke, Antonius J. M.; Matzke, Marjori

2015-01-01

Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate–induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability. PMID:26153075

A human functional protein interaction network and its application to cancer data analysis

PubMed Central

2010-01-01

Background One challenge facing biologists is to tease out useful information from massive data sets for further analysis. A pathway-based analysis may shed light by projecting candidate genes onto protein functional relationship networks. We are building such a pathway-based analysis system. Results We have constructed a protein functional interaction network by extending curated pathways with non-curated sources of information, including protein-protein interactions, gene coexpression, protein domain interaction, Gene Ontology (GO) annotations and text-mined protein interactions, which cover close to 50% of the human proteome. By applying this network to two glioblastoma multiforme (GBM) data sets and projecting cancer candidate genes onto the network, we found that the majority of GBM candidate genes form a cluster and are closer than expected by chance, and the majority of GBM samples have sequence-altered genes in two network modules, one mainly comprising genes whose products are localized in the cytoplasm and plasma membrane, and another comprising gene products in the nucleus. Both modules are highly enriched in known oncogenes, tumor suppressors and genes involved in signal transduction. Similar network patterns were also found in breast, colorectal and pancreatic cancers. Conclusions We have built a highly reliable functional interaction network upon expert-curated pathways and applied this network to the analysis of two genome-wide GBM and several other cancer data sets. The network patterns revealed from our results suggest common mechanisms in the cancer biology. Our system should provide a foundation for a network or pathway-based analysis platform for cancer and other diseases. PMID:20482850

Formin homology 2 domains occur in multiple contexts in angiosperms

PubMed Central

Cvrčková, Fatima; Novotný, Marian; Pícková, Denisa; Žárský, Viktor

2004-01-01

Background Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. Results In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. Conclusions The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity. PMID:15256004

The pilus usher controls protein interactions via domain masking and is functional as an oligomer

DOE PAGES

Werneburg, Glenn T.; Li, Huilin; Henderson, Nadine S.; ...

2015-06-08

The chaperone/usher (CU) pathway is responsible for biogenesis of organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Assembly and secretion of pili by the CU pathway requires a dedicated periplasmic chaperone and a multidomain outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which served as a central switch controlling usher activation. In addition, we demonstratemore » that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.« less

RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

PubMed Central

2014-01-01

Background Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism. PMID:24393626

Heterogeneity in the A33 Protein Impacts the Cross-Protective Efficacy of a Candidate Smallpox DNA Vaccine

DTIC Science & Technology

2008-01-01

include myocarditis, progressive vaccinia, eczema vaccinatum, and even death (Bray, 2003; Cassimatis et al., 2004; Eckart et al., 2004;Kretzschmaret...hundreds of genes, many of which encode for immunomodulatory molecules or molecules with unknown function . The safety risks posed by these molecules...1G10 or function indirectly by impacting the conformation of the physical epitope. However, findings in the literature support the conclusion that

Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein.

PubMed

Chen, Huiqing; Li, Mei; Huang, Guoping; Mai, Weijun; Chen, Keping; Zhou, Yajing

2014-08-01

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

Incorporating molecular and functional context into the analysis and prioritization of human variants associated with cancer

PubMed Central

Peterson, Thomas A; Nehrt, Nathan L; Park, DoHwan

2012-01-01

Background and objective With recent breakthroughs in high-throughput sequencing, identifying deleterious mutations is one of the key challenges for personalized medicine. At the gene and protein level, it has proven difficult to determine the impact of previously unknown variants. A statistical method has been developed to assess the significance of disease mutation clusters on protein domains by incorporating domain functional annotations to assist in the functional characterization of novel variants. Methods Disease mutations aggregated from multiple databases were mapped to domains, and were classified as either cancer- or non-cancer-related. The statistical method for identifying significantly disease-associated domain positions was applied to both sets of mutations and to randomly generated mutation sets for comparison. To leverage the known function of protein domain regions, the method optionally distributes significant scores to associated functional feature positions. Results Most disease mutations are localized within protein domains and display a tendency to cluster at individual domain positions. The method identified significant disease mutation hotspots in both the cancer and non-cancer datasets. The domain significance scores (DS-scores) for cancer form a bimodal distribution with hotspots in oncogenes forming a second peak at higher DS-scores than non-cancer, and hotspots in tumor suppressors have scores more similar to non-cancers. In addition, on an independent mutation benchmarking set, the DS-score method identified mutations known to alter protein function with very high precision. Conclusion By aggregating mutations with known disease association at the domain level, the method was able to discover domain positions enriched with multiple occurrences of deleterious mutations while incorporating relevant functional annotations. The method can be incorporated into translational bioinformatics tools to characterize rare and novel variants within large-scale sequencing studies. PMID:22319177

Dynamics of domain coverage of the protein sequence universe

PubMed Central

2012-01-01

Background The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its “dark matter”. Results Here we suggest that true size of “dark matter” is much larger than stated by current definitions. We propose an approach to reducing the size of “dark matter” by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Conclusions Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of “dark matter”; however, its absolute size increases substantially with the growth of sequence data. PMID:23157439

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

PubMed

Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

2005-07-18

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

Profiling of cellular proteins in porcine reproductive and respiratory syndrome virus virions by proteomics analysis

PubMed Central

2010-01-01

Background Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus, bearing severe economic consequences to the swine industry worldwide. Previous studies on enveloped viruses have shown that many incorporated cellular proteins associated with the virion's membranes that might play important roles in viral infectivity. In this study, we sought to proteomically profile the cellular proteins incorporated into or associated with the virions of a highly virulent PRRSV strain GDBY1, and to provide foundation for further investigations on the roles of incorporated/associated cellular proteins on PRRSV's infectivity. Results In our experiment, sixty one cellular proteins were identified in highly purified PRRSV virions by two-dimensional gel electrophoresis coupled with mass spectrometric approaches. The identified cellular proteins could be grouped into eight functional categories including cytoskeletal proteins, chaperones, macromolecular biosynthesis proteins, metabolism-associated proteins, calcium-dependent membrane-binding proteins and other functional proteins. Among the identified proteins, four have not yet been reported in other studied envelope viruses, namely, guanine nucleotide-binding proteins, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase, peroxiredoxin 1 and galectin-1 protein. The presence of five selected cellular proteins (i.e., β-actin, Tubulin, Annexin A2, heat shock protein Hsp27, and calcium binding proteins S100) in the highly purified PRRSV virions was validated by Western blot and immunogold labeling assays. Conclusions Taken together, the present study has demonstrated the incorporation of cellular proteins in PRRSV virions, which provides valuable information for the further investigations for the effects of individual cellular proteins on the viral replication, assembly, and pathogenesis. PMID:20849641

Host defence related responses in bovine milk during an experimentally induced Streptococcus uberis infection

PubMed Central

2014-01-01

Background Milk contains a range of proteins of moderate or low abundance that contribute to host defence. Characterisation of these proteins, the extent to which their abundance is regulated by pathogenic stimuli, and the variability of their response between and within individual animals would facilitate a better understanding of the molecular basis for this important function of milk. Results We have characterised the host defence proteins in bovine milk and their responses to intra-mammary infection by a common Gram positive mastitis pathogen, Streptococcus uberis, using a combination of 2D gel electrophoresis and GeLC mass spectrometry. In total, 68 host defence-associated proteins were identified, 18 of which have a direct antimicrobial function, 23 of which have a pathogen-recognition function, and 27 of which have a role in modulating inflammatory or immune signalling. The responsiveness of seven proteins was quantified by western blotting; validating the proteomic analyses, quantifying the within- and between animal variability of the responses, and demonstrating the complexity and specificity of the responses to this pathogen. Conclusions These data provide a foundation for understanding the role of milk in host-microbe interaction. Furthermore they provide candidate biomarkers for mastitis diagnosis, and will inform efforts to develop dairy products with improved health-promoting properties. PMID:24721702

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

PubMed Central

Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

2011-01-01

Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

An exploration of alternative visualisations of the basic helix-loop-helix protein interaction network

PubMed Central

Holden, Brian J; Pinney, John W; Lovell, Simon C; Amoutzias, Grigoris D; Robertson, David L

2007-01-01

Background Alternative representations of biochemical networks emphasise different aspects of the data and contribute to the understanding of complex biological systems. In this study we present a variety of automated methods for visualisation of a protein-protein interaction network, using the basic helix-loop-helix (bHLH) family of transcription factors as an example. Results Network representations that arrange nodes (proteins) according to either continuous or discrete information are investigated, revealing the existence of protein sub-families and the retention of interactions following gene duplication events. Methods of network visualisation in conjunction with a phylogenetic tree are presented, highlighting the evolutionary relationships between proteins, and clarifying the context of network hubs and interaction clusters. Finally, an optimisation technique is used to create a three-dimensional layout of the phylogenetic tree upon which the protein-protein interactions may be projected. Conclusion We show that by incorporating secondary genomic, functional or phylogenetic information into network visualisation, it is possible to move beyond simple layout algorithms based on network topology towards more biologically meaningful representations. These new visualisations can give structure to complex networks and will greatly help in interpreting their evolutionary origins and functional implications. Three open source software packages (InterView, TVi and OptiMage) implementing our methods are available. PMID:17683601

How far in-silico computing meets real experiments. A study on the structure and dynamics of spin labeled vinculin tail protein by molecular dynamics simulations and EPR spectroscopy

PubMed Central

2013-01-01

Background Investigation of conformational changes in a protein is a prerequisite to understand its biological function. To explore these conformational changes in proteins we developed a strategy with the combination of molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy. The major goal of this work is to investigate how far computer simulations can meet the experiments. Methods Vinculin tail protein is chosen as a model system as conformational changes within the vinculin protein are believed to be important for its biological function at the sites of cell adhesion. MD simulations were performed on vinculin tail protein both in water and in vacuo environments. EPR experimental data is compared with those of the simulated data for corresponding spin label positions. Results The calculated EPR spectra from MD simulations trajectories of selected spin labelled positions are comparable to experimental EPR spectra. The results show that the information contained in the spin label mobility provides a powerful means of mapping protein folds and their conformational changes. Conclusions The results suggest the localization of dynamic and flexible regions of the vinculin tail protein. This study shows MD simulations can be used as a complementary tool to interpret experimental EPR data. PMID:23445506

The Evolutionary History of Protein Domains Viewed by Species Phylogeny

PubMed Central

Yang, Song; Bourne, Philip E.

2009-01-01

Background Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest. Methodology/Principal Findings A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed. Conclusions/Significance We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species. PMID:20041107

Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins

PubMed Central

Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela

2015-01-01

Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950

COPI selectively drives maturation of the early Golgi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

COPI selectively drives maturation of the early Golgi

DOE PAGES

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham; ...

2015-12-28

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

Local functional descriptors for surface comparison based binding prediction

PubMed Central

2012-01-01

Background Molecular recognition in proteins occurs due to appropriate arrangements of physical, chemical, and geometric properties of an atomic surface. Similar surface regions should create similar binding interfaces. Effective methods for comparing surface regions can be used in identifying similar regions, and to predict interactions without regard to the underlying structural scaffold that creates the surface. Results We present a new descriptor for protein functional surfaces and algorithms for using these descriptors to compare protein surface regions to identify ligand binding interfaces. Our approach uses descriptors of local regions of the surface, and assembles collections of matches to compare larger regions. Our approach uses a variety of physical, chemical, and geometric properties, adaptively weighting these properties as appropriate for different regions of the interface. Our approach builds a classifier based on a training corpus of examples of binding sites of the target ligand. The constructed classifiers can be applied to a query protein providing a probability for each position on the protein that the position is part of a binding interface. We demonstrate the effectiveness of the approach on a number of benchmarks, demonstrating performance that is comparable to the state-of-the-art, with an approach with more generality than these prior methods. Conclusions Local functional descriptors offer a new method for protein surface comparison that is sufficiently flexible to serve in a variety of applications. PMID:23176080

Macrophage differentiation increases expression of the ascorbate transporter (SVCT2)

PubMed Central

Qiao, Huan; May, James M.

2013-01-01

To determine whether macrophage differentiation involves increased uptake of vitamin C, or ascorbic acid, we assessed the expression and function of its transporter SVCT2 during phorbol ester-induced differentiation of human-derived THP-1 monocytes. Induction of THP-1 monocyte differentiation by phorbol 12-myristate 13-acetate (PMA) markedly increased SVCT2 mRNA, protein, and function. When ascorbate was present during PMA-induced differentiation, the increase in SVCT2 protein expression was inhibited, but differentiation was enhanced. PMA-induced SVCT2 protein expression was blocked by inhibitors of protein kinase C (PKC), with most of the affect due to the PKCβI and βII isoforms. Activation of MEK/ERK was sustained up to 48 h after PMA treatment, and the inhibitors completely blocked PMA-stimulated SVCT2 protein expression, indicating an exclusive role for the classical MAP kinase pathway. However, inhibitors of NF-κB activation, NADPH oxidase inhibitors, and several antioxidants also partially prevented SVCT2 induction, suggesting diverse distal routes for control of SVCT2 transcription. Both known promoters for the SVCT2 were involved in these effects. In conclusion, PMA-induced monocyte-macrophage differentiation is enhanced by ascorbate and associated with increased expression and function of the SVCT2 protein through a pathway involving sustained activation of PKCβI/II, MAP kinase, NADPH oxidase, and NF-κB. PMID:19232538

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity

PubMed Central

Mittal, Nimisha; Muthuswami, Rohini

2017-01-01

Background Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. Methodology Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. Conclusion Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis. PMID:28493888

Salivary proteomics of healthy dogs: An in depth catalog

PubMed Central

Furrow, Eva; Souza, Clarissa P.; Granick, Jennifer L.; de Jong, Ebbing P.; Griffin, Timothy J.; Wang, Xiong

2018-01-01

Objective To provide an in-depth catalog of the salivary proteome and endogenous peptidome of healthy dogs, evaluate proteins and peptides with antimicrobial properties, and compare the most common salivary proteins and peptides between different breed phylogeny groups. Methods 36 healthy dogs without evidence of periodontal disease representing four breed phylogeny groups, based upon single nucleotide polymorphism haplotypes (ancient, herding/sighthound, and two miscellaneous groups). Saliva collected from dogs was pooled by phylogeny group and analyzed using nanoscale liquid chromatography-tandem mass spectrometry. Resulting tandem mass spectra were compared to databases for identification of endogenous peptides and inferred proteins. Results 2,491 proteins and endogenous peptides were found in the saliva of healthy dogs with no periodontal disease. All dog phylogeny groups’ saliva was rich in proteins and peptides with antimicrobial functions. The ancient breeds group was distinct in that it contained unique proteins and was missing many proteins and peptides present in the other groups. Conclusions and clinical relevance Using a sophisticated nanoscale liquid chromatography-tandem mass spectrometry, we were able to identify 10-fold more salivary proteins than previously reported in dogs. Seven of the top 10 most abundant proteins or peptides serve immune functions and many more with various antimicrobial mechanisms were found. This is the most comprehensive analysis of healthy canine saliva to date, and will provide the groundwork for future studies analyzing salivary proteins and endogenous peptides in disease states. PMID:29329347

RGS2 is a feedback inhibitor of melatonin production in the pineal gland

PubMed Central

Matsuo, Masahiro; Coon, Steven L.; Klein, David C.

2014-01-01

The 24-h rhythmic production of melatonin by the pineal gland is essential for coordinating circadian physiology. Melatonin production increases at night in response to the release of norepinephrine from sympathetic nerve processes which innervate the pineal gland. This signal is transduced through G-protein-coupled adrenergic receptors. Here, we found that the abundance of regulator of G-protein signaling 2 (RGS2) increases at night, that expression is increased by norepinephrine and that this protein has a negative feedback effect on melatonin production. These data are consistent with the conclusion that RGS2 functions on a daily basis to negatively modulate melatonin production. PMID:23523917

A genetic replacement system for selection-based engineering of essential proteins

PubMed Central

2012-01-01

Background Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. Results We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. Conclusions We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies. PMID:22898007

Functional analysis of a regulator of G-protein signaling CgRGS1 in the rubber tree anthracnose fungus Colletotrichum gloeosporioides.

PubMed

Liu, Zhi-Qiang; Wu, Man-Li; Ke, Zhi-Jian; Liu, Wen-Bo; Li, Xiao-Yu

2018-04-01

Colletotrichum gloeosporioides is the causal agent of rubber anthracnose, which is also one of the important biological factors threatening the development of natural rubber industry in the world. Regulators of G-protein signaling (RGS) are key negative regulators of G-proteins, which play important roles in growth, development and pathogenic processes of plant pathogens. In this study, a RGS gene CgRGS1 was functionally characterized in C. gloeosporioides. Compared to the wild type, the CgRGS1 deletion mutant had slow vegetative growth, reduced conidia with multi-end germination, low appressorium formation rate, high resistance to oxidative stress and SDS. Moreover, the mutant was sensitive to osmotic pressure and showed decreased virulence. In conclusion, CgRGS1 is involved in regulation of vegetative growth, conidiation, germination, appressorium formation, oxidative stress, osmotic pressure response and pathogenicity in C. gloeosporioides.

The ATM protein kinase and cellular redox signaling: beyond the DNA damage response

PubMed Central

Ditch, Scott; Paull, Tanya T.

2011-01-01

The ataxia-telangiectasia mutated (ATM) protein kinase is best known for its role in the DNA damage response, but recent findings suggest that it also functions as a redox sensor that controls the levels of reactive oxygen species in human cells. Here, we review the evidence supporting the conclusion that ATM can be directly activated by oxidation, as well as various observations from ATM-deficient patients and mouse models that point toward the importance of ATM in oxidative stress responses. We also discuss the roles of this kinase in regulating mitochondrial function and metabolic control through its action on tumor suppressor p53, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 (HIF-1), and how the regulation of these enzymes may be affected in ATM-deficient patients and in cancer cells. PMID:22079189

The ATM protein kinase and cellular redox signaling: beyond the DNA damage response.

PubMed

Ditch, Scott; Paull, Tanya T

2012-01-01

The ataxia-telangiectasia mutated (ATM) protein kinase is best known for its role in the DNA damage response, but recent findings suggest that it also functions as a redox sensor that controls the levels of reactive oxygen species in human cells. Here, we review evidence supporting the conclusion that ATM can be directly activated by oxidation, as well as various observations from ATM-deficient patients and mouse models that point to the importance of ATM in oxidative stress responses. We also discuss the roles of this kinase in regulating mitochondrial function and metabolic control through its action on tumor suppressor p53, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor 1 (HIF1), and how the regulation of these enzymes may be affected in ATM-deficient patients and in cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

NASA Astrophysics Data System (ADS)

Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

2017-03-01

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

NASA Astrophysics Data System (ADS)

Encarnación, Medina-Carmona; Palomino-Morales, Rogelio J.; Fuchs, Julian E.; Esperanza, Padín-Gonzalez; Noel, Mesa-Torres; Salido, Eduardo; Timson, David J.; Pey, Angel L.

2016-02-01

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.

An integrative approach to inferring biologically meaningful gene modules

PubMed Central

2011-01-01

Background The ability to construct biologically meaningful gene networks and modules is critical for contemporary systems biology. Though recent studies have demonstrated the power of using gene modules to shed light on the functioning of complex biological systems, most modules in these networks have shown little association with meaningful biological function. We have devised a method which directly incorporates gene ontology (GO) annotation in construction of gene modules in order to gain better functional association. Results We have devised a method, Semantic Similarity-Integrated approach for Modularization (SSIM) that integrates various gene-gene pairwise similarity values, including information obtained from gene expression, protein-protein interactions and GO annotations, in the construction of modules using affinity propagation clustering. We demonstrated the performance of the proposed method using data from two complex biological responses: 1. the osmotic shock response in Saccharomyces cerevisiae, and 2. the prion-induced pathogenic mouse model. In comparison with two previously reported algorithms, modules identified by SSIM showed significantly stronger association with biological functions. Conclusions The incorporation of semantic similarity based on GO annotation with gene expression and protein-protein interaction data can greatly enhance the functional relevance of inferred gene modules. In addition, the SSIM approach can also reveal the hierarchical structure of gene modules to gain a broader functional view of the biological system. Hence, the proposed method can facilitate comprehensive and in-depth analysis of high throughput experimental data at the gene network level. PMID:21791051

Down-Regulating γ-Gliadins in Bread Wheat Leads to Non-Specific Increases in Other Gluten Proteins and Has No Major Effect on Dough Gluten Strength

PubMed Central

Pistón, Fernando; Gil-Humanes, Javier; Rodríguez-Quijano, Marta; Barro, Francisco

2011-01-01

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins. PMID:21935456

Predicting Drug-Target Interaction Networks Based on Functional Groups and Biological Features

PubMed Central

Shi, Xiao-He; Hu, Le-Le; Kong, Xiangyin; Cai, Yu-Dong; Chou, Kuo-Chen

2010-01-01

Background Study of drug-target interaction networks is an important topic for drug development. It is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. As a complement, the in silico prediction methods can provide us with very useful information in a timely manner. Methods/Principal Findings To realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. The optimal feature selection procedures are adopted by means of the mRMR (Maximum Relevance Minimum Redundancy) method. Instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, G-protein- coupled receptors and nuclear receptors. Thus, four independent predictors are established using the Nearest Neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. As a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are 85.48%, 80.78%, 78.49%, and 85.66%, respectively. Conclusion/Significance Our results indicate that the network prediction system thus established is quite promising and encouraging. PMID:20300175

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

PubMed Central

Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

2012-01-01

Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins. PMID:22359651

Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein

PubMed Central

Xu, Han; Wang, Tao; Zhang, Yanming

2015-01-01

Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn. PMID:26035722

Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity

PubMed Central

Westernberg, Luise; Pham, John; Lane, Jerome; Paul, Sinu; Greenbaum, Jason; Stranzl, Thomas; Lund, Gitte; Hoof, Ilka; Holm, Jens; Würtzen, Peter A; Meno, Kåre H.; Frazier, April; Schulten, Veronique; Andersen, Peter S.; Peters, Bjoern; Sette, Alessandro

2016-01-01

BACKGROUND House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking. OBJECTIVE To comprehensively analyze the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function. METHODS Proteomic analysis (liquid chromatography tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity and putative function analyses were performed in silico according to gene ontology (GO) annotations. RESULTS Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE-reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T-cell response, underlining the heterogeneity of T cell responses to HDM allergens. CONCLUSIONS AND CLINICAL RELEVANCE Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM. PMID:27684489

Deep Illumina-Based Shotgun Sequencing Reveals Dietary Effects on the Structure and Function of the Fecal Microbiome of Growing Kittens

PubMed Central

Deusch, Oliver; O’Flynn, Ciaran; Colyer, Alison; Morris, Penelope; Allaway, David; Jones, Paul G.; Swanson, Kelly S.

2014-01-01

Background Previously, we demonstrated that dietary protein:carbohydrate ratio dramatically affects the fecal microbial taxonomic structure of kittens using targeted 16S gene sequencing. The present study, using the same fecal samples, applied deep Illumina shotgun sequencing to identify the diet-associated functional potential and analyze taxonomic changes of the feline fecal microbiome. Methodology & Principal Findings Fecal samples from kittens fed one of two diets differing in protein and carbohydrate content (high–protein, low–carbohydrate, HPLC; and moderate-protein, moderate-carbohydrate, MPMC) were collected at 8, 12 and 16 weeks of age (n = 6 per group). A total of 345.3 gigabases of sequence were generated from 36 samples, with 99.75% of annotated sequences identified as bacterial. At the genus level, 26% and 39% of reads were annotated for HPLC- and MPMC-fed kittens, with HPLC-fed cats showing greater species richness and microbial diversity. Two phyla, ten families and fifteen genera were responsible for more than 80% of the sequences at each taxonomic level for both diet groups, consistent with the previous taxonomic study. Significantly different abundances between diet groups were observed for 324 genera (56% of all genera identified) demonstrating widespread diet-induced changes in microbial taxonomic structure. Diversity was not affected over time. Functional analysis identified 2,013 putative enzyme function groups were different (p<0.000007) between the two dietary groups and were associated to 194 pathways, which formed five discrete clusters based on average relative abundance. Of those, ten contained more (p<0.022) enzyme functions with significant diet effects than expected by chance. Six pathways were related to amino acid biosynthesis and metabolism linking changes in dietary protein with functional differences of the gut microbiome. Conclusions These data indicate that feline feces-derived microbiomes have large structural and functional differences relating to the dietary protein:carbohydrate ratio and highlight the impact of diet early in life. PMID:25010839

Emergence of novel domains in proteins

PubMed Central

2013-01-01

Background Proteins are composed of a combination of discrete, well-defined, sequence domains, associated with specific functions that have arisen at different times during evolutionary history. The emergence of novel domains is related to protein functional diversification and adaptation. But currently little is known about how novel domains arise and how they subsequently evolve. Results To gain insights into the impact of recently emerged domains in protein evolution we have identified all human young protein domains that have emerged in approximately the past 550 million years. We have classified them into vertebrate-specific and mammalian-specific groups, and compared them to older domains. We have found 426 different annotated young domains, totalling 995 domain occurrences, which represent about 12.3% of all human domains. We have observed that 61.3% of them arose in newly formed genes, while the remaining 38.7% are found combined with older domains, and have very likely emerged in the context of a previously existing protein. Young domains are preferentially located at the N-terminus of the protein, indicating that, at least in vertebrates, novel functional sequences often emerge there. Furthermore, young domains show significantly higher non-synonymous to synonymous substitution rates than older domains using human and mouse orthologous sequence comparisons. This is also true when we compare young and old domains located in the same protein, suggesting that recently arisen domains tend to evolve in a less constrained manner than older domains. Conclusions We conclude that proteins tend to gain domains over time, becoming progressively longer. We show that many proteins are made of domains of different age, and that the fastest evolving parts correspond to the domains that have been acquired more recently. PMID:23425224

Effect of a High-Protein Diet on Kidney Function in Healthy Adults: Results From the OmniHeart Trial

PubMed Central

Juraschek, Stephen P.; Appel, Lawrence J.; Anderson, Cheryl A.M.; Miller, Edgar R.

2013-01-01

Background Consumption of a diet high in protein can cause glomerular hyperfiltration, a potentially maladaptive response, which may accelerate the progression of kidney disease. Study Design An ancillary study of the OmniHeart trial, a randomized 3-period crossover feeding trial testing the effects of partial replacement of carbohydrate with protein on kidney function. Setting & Participants Healthy adults (N=164) with prehypertension or stage 1 hypertension at a community-based research clinic with a metabolic kitchen. Intervention Participants were fed each of 3 diets for 6 weeks. Feeding periods were separated by a 2- to 4-week washout period. Weight was held constant on each diet. The 3 diets emphasized carbohydrate, protein, or unsaturated fat; dietary protein was either 15% (carbohydrate and unsaturated fat diets) or 25% (protein diet) of energy intake. Outcomes Fasting serum creatinine, cystatin C, and β2-microglobulin levels, estimated glomerular filtration rate (eGFR). Measurements Serum creatinine, cystatin C, and β2-microglobulin collected at the end of each feeding period. Results Baseline cystatin C-based eGFR was 92.0±16.3 (SD) mL/min/1.73 m2. Compared with the carbohydrate and unsaturated fat diets, the protein diet increased cystatin C-based eGFR by ~4 mL/min/1.73 m2 (P < 0.001). The effects of the protein diet on kidney function were independent of changes in blood pressure. There was no significant difference between the carbohydrate and unsaturated fat diets. Limitations Participants did not have kidney disease at baseline. Conclusions A healthy diet rich in protein increased eGFR. Whether long-term consumption of a high-protein diet leads to kidney disease is uncertain. PMID:23219108

Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase

PubMed Central

2014-01-01

Background Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism. Results BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications. Conclusions Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively. PMID:24742328

Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Ancrum, Tiffany; Manevich, Yefim; Townsend, Danyelle M.

2017-01-01

Abstract Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2−/−) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2−/− cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Innovation: Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Conclusions: Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR through S-glutathionylation of a series of key interrelated proteins. Antioxid. Redox Signal. 26, 247–261. PMID:26838680

Exploring representations of protein structure for automated remote homology detection and mapping of protein structure space

PubMed Central

2014-01-01

Background Due to rapid sequencing of genomes, there are now millions of deposited protein sequences with no known function. Fast sequence-based comparisons allow detecting close homologs for a protein of interest to transfer functional information from the homologs to the given protein. Sequence-based comparison cannot detect remote homologs, in which evolution has adjusted the sequence while largely preserving structure. Structure-based comparisons can detect remote homologs but most methods for doing so are too expensive to apply at a large scale over structural databases of proteins. Recently, fragment-based structural representations have been proposed that allow fast detection of remote homologs with reasonable accuracy. These representations have also been used to obtain linearly-reducible maps of protein structure space. It has been shown, as additionally supported from analysis in this paper that such maps preserve functional co-localization of the protein structure space. Methods Inspired by a recent application of the Latent Dirichlet Allocation (LDA) model for conducting structural comparisons of proteins, we propose higher-order LDA-obtained topic-based representations of protein structures to provide an alternative route for remote homology detection and organization of the protein structure space in few dimensions. Various techniques based on natural language processing are proposed and employed to aid the analysis of topics in the protein structure domain. Results We show that a topic-based representation is just as effective as a fragment-based one at automated detection of remote homologs and organization of protein structure space. We conduct a detailed analysis of the information content in the topic-based representation, showing that topics have semantic meaning. The fragment-based and topic-based representations are also shown to allow prediction of superfamily membership. Conclusions This work opens exciting venues in designing novel representations to extract information about protein structures, as well as organizing and mining protein structure space with mature text mining tools. PMID:25080993

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

PubMed Central

2013-01-01

Background The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. Results and discussion Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. Conclusions Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. PMID:23442797

Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populus and the plant kingdom

PubMed Central

2013-01-01

Background Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. Results We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. Conclusion In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined. PMID:23957885

SoxNeuro orchestrates central nervous system specification and differentiation in Drosophila and is only partially redundant with Dichaete

PubMed Central

2014-01-01

Background Sox proteins encompass an evolutionarily conserved family of transcription factors with critical roles in animal development and stem cell biology. In common with vertebrates, the Drosophila group B proteins SoxNeuro and Dichaete are involved in central nervous system development, where they play both similar and unique roles in gene regulation. Sox genes show extensive functional redundancy across metazoans, but the molecular basis underpinning functional compensation mechanisms at the genomic level are currently unknown. Results Using a combination of genome-wide binding analysis and gene expression profiling, we show that SoxNeuro directs embryonic neural development from the early specification of neuroblasts through to the terminal differentiation of neurons and glia. To address the issue of functional redundancy and compensation at a genomic level, we compare SoxNeuro and Dichaete binding, identifying common and independent binding events in wild-type conditions, as well as instances of compensation and loss of binding in mutant backgrounds. Conclusions We find that early aspects of group B Sox functions in the central nervous system, such as stem cell maintenance and dorsoventral patterning, are highly conserved. However, in contrast to vertebrates, we find that Drosophila group B1 proteins also play prominent roles during later aspects of neural morphogenesis. Our analysis of the functional relationship between SoxNeuro and Dichaete uncovers evidence for redundant and independent functions for each protein, along with unexpected examples of compensation and interdependency, thus providing new insights into the general issue of transcription factor functional redundancy. PMID:24886562

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

PubMed Central

2012-01-01

Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution. PMID:23148531

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck

PubMed Central

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-01-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level. PMID:21841170

Protein Expression Profile of Twenty-Week-Old Diabetic db/db and Non-Diabetic Mice Livers: A Proteomic and Bioinformatic Analysis.

PubMed

Guzmán-Flores, Juan Manuel; Flores-Pérez, Elsa Cristina; Hernández-Ortiz, Magdalena; Vargas-Ortiz, Katya; Ramírez-Emiliano, Joel; Encarnación-Guevara, Sergio; Pérez-Vázquez, Victoriano

2018-06-01

Type 2 diabetes mellitus is characterized by insulin resistance in the liver. Insulin is not only involved in carbohydrate metabolism, it also regulates protein synthesis. This work describes the expression of proteins in the liver of a diabetic mouse and identifies the metabolic pathways involved. Twenty-week-old diabetic db/db mice were hepatectomized, after which proteins were separated by 2D-Polyacrylamide Gel Electrophoresis (2D-PAGE). Spots varying in intensity were analyzed using mass spectrometry, and biological function was assigned by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software. A differential expression of 26 proteins was identified; among these were arginase-1, pyruvate carboxylase, peroxiredoxin-1, regucalcin, and sorbitol dehydrogenase. Bioinformatics analysis indicated that many of these proteins are mitochondrial and participate in metabolic pathways, such as the citrate cycle, the fructose and mannose metabolism, and glycolysis or gluconeogenesis. In addition, these proteins are related to oxidation⁻reduction reactions and molecular function of vitamin binding and amino acid metabolism. In conclusion, the proteomic profile of the liver of diabetic mouse db/db exhibited mainly alterations in the metabolism of carbohydrates and nitrogen. These differences illustrate the heterogeneity of diabetes in its different stages and under different conditions and highlights the need to improve treatments for this disease.

Probiotics promote endocytic allergen degradation in gut epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Chun-Hua; Liu, Zhi-Qiang; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON

Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barriermore » function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.« less

Improved packing of protein side chains with parallel ant colonies

PubMed Central

2014-01-01

Introduction The accurate packing of protein side chains is important for many computational biology problems, such as ab initio protein structure prediction, homology modelling, and protein design and ligand docking applications. Many of existing solutions are modelled as a computational optimisation problem. As well as the design of search algorithms, most solutions suffer from an inaccurate energy function for judging whether a prediction is good or bad. Even if the search has found the lowest energy, there is no certainty of obtaining the protein structures with correct side chains. Methods We present a side-chain modelling method, pacoPacker, which uses a parallel ant colony optimisation strategy based on sharing a single pheromone matrix. This parallel approach combines different sources of energy functions and generates protein side-chain conformations with the lowest energies jointly determined by the various energy functions. We further optimised the selected rotamers to construct subrotamer by rotamer minimisation, which reasonably improved the discreteness of the rotamer library. Results We focused on improving the accuracy of side-chain conformation prediction. For a testing set of 442 proteins, 87.19% of X1 and 77.11% of X12 angles were predicted correctly within 40° of the X-ray positions. We compared the accuracy of pacoPacker with state-of-the-art methods, such as CIS-RR and SCWRL4. We analysed the results from different perspectives, in terms of protein chain and individual residues. In this comprehensive benchmark testing, 51.5% of proteins within a length of 400 amino acids predicted by pacoPacker were superior to the results of CIS-RR and SCWRL4 simultaneously. Finally, we also showed the advantage of using the subrotamers strategy. All results confirmed that our parallel approach is competitive to state-of-the-art solutions for packing side chains. Conclusions This parallel approach combines various sources of searching intelligence and energy functions to pack protein side chains. It provides a frame-work for combining different inaccuracy/usefulness objective functions by designing parallel heuristic search algorithms. PMID:25474164

Isolation of anticancer drug TAXOL from Pestalotiopsis breviseta with apoptosis and B-Cell lymphoma protein docking studies

PubMed Central

Kathiravan, G.; Sureban, Sripathi M.; Sree, Harsha N.; Bhuvaneshwari, V.; Kramony, Evelin

2012-01-01

Background: Extraction and investigation of TAXOL from Pestalotiopsis breviseta (Sacc.) using protein docking, which is a computational technique that samples conformations of small molecules in protein-binding sites. Scoring functions are used to assess which of these conformations best complements the protein binding site and active site prediction. Materials and Methods: Coelomycetous fungi P. breviseta (Sacc.) Steyaert was screened for the production of TAXOL, an anticancer drug. Results: TAXOL production was confirmed by the following methods: Ultraviolet (UV) spectroscopic analysis, Infrared analysis, High performance liquid chromatography analysis (HPLC), and Liquid chromatography mass spectrum (LC-MASS). TAXOL produced by the fungi was compared with authentic TAXOL, and protein docking studies were performed. Conclusion: The BCL2 protein of human origin showed a higher affinity toward the compound paclitaxel. It has the binding energy value of −13.0061 (KJ/Mol) with four hydrogen bonds. PMID:24808664

Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway.

PubMed

Park, Arnold; Yun, Tatyana; Vigant, Frederic; Pernet, Olivier; Won, Sohui T; Dawes, Brian E; Bartkowski, Wojciech; Freiberg, Alexander N; Lee, Benhur

2016-05-01

The budding of Nipah virus, a deadly member of the Henipavirus genus within the Paramyxoviridae, has been thought to be independent of the host ESCRT pathway, which is critical for the budding of many enveloped viruses. This conclusion was based on the budding properties of the virus matrix protein in the absence of other virus components. Here, we find that the virus C protein, which was previously investigated for its role in antagonism of innate immunity, recruits the ESCRT pathway to promote efficient virus release. Inhibition of ESCRT or depletion of the ESCRT factor Tsg101 abrogates the C enhancement of matrix budding and impairs live Nipah virus release. Further, despite the low sequence homology of the C proteins of known henipaviruses, they all enhance the budding of their cognate matrix proteins, suggesting a conserved and previously unknown function for the henipavirus C proteins.

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

PubMed Central

Mills, Ian G.; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E.

2005-01-01

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription. PMID:16027218

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

PubMed Central

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O.; Schnölzer, Martina

2010-01-01

Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades. PMID:20224743

Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.

PubMed

Doronin, Konstantin; Toth, Karoly; Kuppuswamy, Mohan; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

2003-01-20

Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.

CCN proteins: A centralized communication network.

PubMed

Perbal, Bernard

2013-08-01

The CCN family of proteins includes six members presently known as CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6. These proteins were originally designated CYR61, CTGF, NOV, and WISP-1, WISP-2, WISP-3. Although these proteins share a significant amount of structural features and a partial identity with other large families of regulatory proteins, they exhibit different biological functions. A critical examination of the progress made over the past two decades, since the first CCN proteins were discovered brings me to the conclusion that most of our present knowledge regarding the functions of these proteins was predicted very early after their discovery. In an effort to point out some of the gaps that prevent us to reach a comprehensive view of the functional interactions between CCN proteins, it is necessary to reconsider carefully data that was already published and put aside, either because the scientific community was not ready to accept them, or because they were not fitting with the « consensus » when they were published. This review article points to avenues that were not attracting the attention that they deserved. However, it is quite obvious that the six members of this unique family of tetra-modular proteins must act in concert, either simultaneously or sequentially, on the same sites or at different times in the life of living organisms. A better understanding of the spatio-temporal regulation of CCN proteins expression requires considering the family as such, not as a set of single proteins related only by their name. As proposed in this review, there is enough convincing pieces of evidence, at the present time, in favor of these proteins playing a role in the coordination of multiple signaling pathways, and constituting a Centralized Communication Network. Deciphering the hierarchy of regulatory circuits involved in this complex system is an important challenge for the near future. In this article, I would like to briefly review the concept of a CCN family of proteins and critically examine the progress made over the past 10 years in the understanding of their biological functions and involvement in both normal and pathological processes.

Clearing the skies over modular polyketide synthases.

PubMed

Sherman, David H; Smith, Janet L

2006-09-19

Modular polyketide synthases (PKSs) are large multifunctional proteins that synthesize complex polyketide metabolites in microbial cells. A series of recent studies confirm the close protein structural relationship between catalytic domains in the type I mammalian fatty acid synthase (FAS) and the basic synthase unit of the modular PKS. They also establish a remarkable similarity in the overall organization of the type I FAS and the PKS module. This information provides important new conclusions about catalytic domain architecture, function, and molecular recognition that are essential for future efforts to engineer useful polyketide metabolites with valuable biological activities.

Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines

PubMed Central

2013-01-01

Background Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. Methods To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Results Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. Conclusions Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases. PMID:23316740

The extracellular Leucine-Rich Repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns

PubMed Central

Dolan, Jackie; Walshe, Karen; Alsbury, Samantha; Hokamp, Karsten; O'Keeffe, Sean; Okafuji, Tatsuya; Miller, Suzanne FC; Tear, Guy; Mitchell, Kevin J

2007-01-01

Background Leucine-rich repeats (LRRs) are highly versatile and evolvable protein-ligand interaction motifs found in a large number of proteins with diverse functions, including innate immunity and nervous system development. Here we catalogue all of the extracellular LRR (eLRR) proteins in worms, flies, mice and humans. We use convergent evidence from several transmembrane-prediction and motif-detection programs, including a customised algorithm, LRRscan, to identify eLRR proteins, and a hierarchical clustering method based on TribeMCL to establish their evolutionary relationships. Results This yields a total of 369 proteins (29 in worm, 66 in fly, 135 in mouse and 139 in human), many of them of unknown function. We group eLRR proteins into several classes: those with only LRRs, those that cluster with Toll-like receptors (Tlrs), those with immunoglobulin or fibronectin-type 3 (FN3) domains and those with some other domain. These groups show differential patterns of expansion and diversification across species. Our analyses reveal several clusters of novel genes, including two Elfn genes, encoding transmembrane proteins with eLRRs and an FN3 domain, and six genes encoding transmembrane proteins with eLRRs only (the Elron cluster). Many of these are expressed in discrete patterns in the developing mouse brain, notably in the thalamus and cortex. We have also identified a number of novel fly eLRR proteins with discrete expression in the embryonic nervous system. Conclusion This study provides the necessary foundation for a systematic analysis of the functions of this class of genes, which are likely to include prominently innate immunity, inflammation and neural development, especially the specification of neuronal connectivity. PMID:17868438

Effectiveness of exercise and protein supplementation intervention on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia.

PubMed

Shahar, Suzana; Kamaruddin, Norshafarina Shari; Badrasawi, Manal; Sakian, Noor Ibrahim Mohamed; Abd Manaf, Zahara; Yassin, Zaitun; Joseph, Leonard

2013-01-01

Sarcopenia, characterized as muscle loss that occurs with aging, is a major health problem in an aging population, due to its implications on mobility, quality of life, and fall risk. Protein supplementation could improve the physical fitness by increasing protein anabolism, and exercise has a documented evidence of positive effect on functional status among the elderly. However, the combined effect of both protein supplementation and exercise has not been investigated among sarcopenic elderly in the Asian population. Thus, this study aimed to determine the effectiveness of exercise intervention and protein supplementation either alone or in combination for 12 weeks, on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia. Sixty five sarcopenic elderly Malays aged 60-74 years were assigned to the control group, exercise group (ExG), protein supplementation group (PrG), or the combination of exercise and protein supplementation group. A significant interaction effect between body weight and body mass index (BMI) was observed, with the PrG (-2.1% body weight, -1.8% BMI) showing the highest reductions. Further, there was a decrease in % body fat (-4.5%) and an increase in fat-free mass (kg) (+5.7%) in the ExG after 12 weeks (P < 0.05). The highest increments in lower and upper body strength were observed in the PrG (73.2%) and ExG (47.6%), respectively. In addition, the ExG showed a reduction in superoxide dismutase (SOD) levels, and both interventions did not alter either lipid or protein oxidation. In conclusion, the exercise program was found to improve muscle strength and body composition, while protein supplementation reduced body weight and increased upper body strength, among sarcopenic elderly in Malaysia.

Effectiveness of exercise and protein supplementation intervention on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia

PubMed Central

Shahar, Suzana; Kamaruddin, Norshafarina Shari; Badrasawi, Manal; Sakian, Noor Ibrahim Mohamed; Manaf, Zahara Abd; Yassin, Zaitun; Joseph, Leonard

2013-01-01

Sarcopenia, characterized as muscle loss that occurs with aging, is a major health problem in an aging population, due to its implications on mobility, quality of life, and fall risk. Protein supplementation could improve the physical fitness by increasing protein anabolism, and exercise has a documented evidence of positive effect on functional status among the elderly. However, the combined effect of both protein supplementation and exercise has not been investigated among sarcopenic elderly in the Asian population. Thus, this study aimed to determine the effectiveness of exercise intervention and protein supplementation either alone or in combination for 12 weeks, on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia. Sixty five sarcopenic elderly Malays aged 60–74 years were assigned to the control group, exercise group (ExG), protein supplementation group (PrG), or the combination of exercise and protein supplementation group. A significant interaction effect between body weight and body mass index (BMI) was observed, with the PrG (−2.1% body weight, −1.8% BMI) showing the highest reductions. Further, there was a decrease in % body fat (−4.5%) and an increase in fat-free mass (kg) (+5.7%) in the ExG after 12 weeks (P < 0.05). The highest increments in lower and upper body strength were observed in the PrG (73.2%) and ExG (47.6%), respectively. In addition, the ExG showed a reduction in superoxide dismutase (SOD) levels, and both interventions did not alter either lipid or protein oxidation. In conclusion, the exercise program was found to improve muscle strength and body composition, while protein supplementation reduced body weight and increased upper body strength, among sarcopenic elderly in Malaysia. PMID:24143082

The role of nonbonded interactions in the conformational dynamics of organophosphorous hydrolase adsorbed onto functionalized mesoporous silica surfaces.

PubMed

Gomes, Diego E B; Lins, Roberto D; Pascutti, Pedro G; Lei, Chenghong; Soares, Thereza A

2010-01-14

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein

PubMed Central

2014-01-01

Background Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. Results We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. Conclusion CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions. PMID:24885367

Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

PubMed

Farach-Carson, Mary C; Warren, Curtis R; Harrington, Daniel A; Carson, Daniel D

2014-02-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. © 2013.

Border Patrol: Insights into the Unique Role of Perlecan/Heparan Sulfate Proteoglycan2 at Cell and Tissue Borders

PubMed Central

Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel A.; Carson, Daniel D.

2013-01-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550M years) extracellular matrix molecules. In vertebrates, perlecan’s five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. PMID:24001398

Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL): adapting the Partial Phylogenetic Profiling algorithm to scan sequences for signatures that predict protein function

PubMed Central

2010-01-01

Background Comparative genomics methods such as phylogenetic profiling can mine powerful inferences from inherently noisy biological data sets. We introduce Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL), a method that applies the Partial Phylogenetic Profiling (PPP) approach locally within a protein sequence to discover short sequence signatures associated with functional sites. The approach is based on the basic scoring mechanism employed by PPP, namely the use of binomial distribution statistics to optimize sequence similarity cutoffs during searches of partitioned training sets. Results Here we illustrate and validate the ability of the SIMBAL method to find functionally relevant short sequence signatures by application to two well-characterized protein families. In the first example, we partitioned a family of ABC permeases using a metabolic background property (urea utilization). Thus, the TRUE set for this family comprised members whose genome of origin encoded a urea utilization system. By moving a sliding window across the sequence of a permease, and searching each subsequence in turn against the full set of partitioned proteins, the method found which local sequence signatures best correlated with the urea utilization trait. Mapping of SIMBAL "hot spots" onto crystal structures of homologous permeases reveals that the significant sites are gating determinants on the cytosolic face rather than, say, docking sites for the substrate-binding protein on the extracellular face. In the second example, we partitioned a protein methyltransferase family using gene proximity as a criterion. In this case, the TRUE set comprised those methyltransferases encoded near the gene for the substrate RF-1. SIMBAL identifies sequence regions that map onto the substrate-binding interface while ignoring regions involved in the methyltransferase reaction mechanism in general. Neither method for training set construction requires any prior experimental characterization. Conclusions SIMBAL shows that, in functionally divergent protein families, selected short sequences often significantly outperform their full-length parent sequence for making functional predictions by sequence similarity, suggesting avenues for improved functional classifiers. When combined with structural data, SIMBAL affords the ability to localize and model functional sites. PMID:20102603

The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains

PubMed Central

Iyer, Lakshminarayan M; Burroughs, A Maxwell; Aravind, L

2006-01-01

Background Ubiquitin (Ub)-mediated signaling is one of the hallmarks of all eukaryotes. Prokaryotic homologs of Ub (ThiS and MoaD) and E1 ligases have been studied in relation to sulfur incorporation reactions in thiamine and molybdenum/tungsten cofactor biosynthesis. However, there is no evidence for entire protein modification systems with Ub-like proteins and deconjugation by deubiquitinating enzymes in prokaryotes. Hence, the evolutionary assembly of the eukaryotic Ub-signaling apparatus remains unclear. Results We systematically analyzed prokaryotic Ub-related β-grasp fold proteins using sensitive sequence profile searches and structural analysis. Consequently, we identified novel Ub-related proteins beyond the characterized ThiS, MoaD, TGS, and YukD domains. To understand their functional associations, we sought and recovered several conserved gene neighborhoods and domain architectures. These included novel associations involving diverse sulfur metabolism proteins, siderophore biosynthesis and the gene encoding the transfer mRNA binding protein SmpB, as well as domain fusions between Ub-like domains and PIN-domain related RNAses. Most strikingly, we found conserved gene neighborhoods in phylogenetically diverse bacteria combining genes for JAB domains (the primary de-ubiquitinating isopeptidases of the proteasomal complex), along with E1-like adenylating enzymes and different Ub-related proteins. Further sequence analysis of other conserved genes in these neighborhoods revealed several Ub-conjugating enzyme/E2-ligase related proteins. Genes for an Ub-like protein and a JAB domain peptidase were also found in the tail assembly gene cluster of certain caudate bacteriophages. Conclusion These observations imply that members of the Ub family had already formed strong functional associations with E1-like proteins, UBC/E2-related proteins, and JAB peptidases in the bacteria. Several of these Ub-like proteins and the associated protein families are likely to function together in signaling systems just as in eukaryotes. PMID:16859499

Oxidative Stress and Huntington's Disease: The Good, The Bad, and The Ugly.

PubMed

Kumar, Amit; Ratan, Rajiv R

2016-10-01

Redox homeostasis is crucial for proper cellular functions, including receptor tyrosine kinase signaling, protein folding, and xenobiotic detoxification. Under basal conditions, there is a balance between oxidants and antioxidants. This balance facilitates the ability of oxidants, such as reactive oxygen species, to play critical regulatory functions through a direct modification of a small number of amino acids (e.g. cysteine) on signaling proteins. These signaling functions leverage tight spatial, amplitude, and temporal control of oxidant concentrations. However, when oxidants overwhelm the antioxidant capacity, they lead to a harmful condition of oxidative stress. Oxidative stress has long been held to be one of the key players in disease progression for Huntington's disease (HD). In this review, we will critically review this evidence, drawing some intermediate conclusions, and ultimately provide a framework for thinking about the role of oxidative stress in the pathophysiology of HD.

Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

PubMed Central

Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

2012-01-01

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

PubMed Central

Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; van den Akker, Peter C; Pasmooij, Anna MG; Nyström, Alexander

2016-01-01

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)—a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB. PMID:27157667

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

PubMed Central

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

2012-01-01

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer. PMID:22754528

BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

PubMed Central

Kuo, P-C; Huang, C-W; Lee, C-I; Chang, H-W; Hsieh, S-W; Chung, Y-P; Lee, M-S; Huang, C-S; Tsao, L-P; Tsao, Y-P; Chen, S-L

2015-01-01

Background: We showed previously that breast carcinoma amplified sequence 2 (BCAS2) functions as a negative regulator of p53. We also found that BCAS2 is a potential AR-associated protein. AR is essential for the growth and survival of prostate carcinoma. Therefore we characterised the correlation between BCAS2 and AR. Methods: Protein interactions were examined by GST pull-down assay and co-immunoprecipitation. Clinical prostate cancer (PCa) specimens were evaluated by immunohistochemical assay. AR transcriptional activity and LNCaP cell growth were assessed by luciferase assay and MTT assay, respectively. Results: BCAS2 expression was significantly increased in PCa. BCAS2 stabilised AR protein through both hormone-dependent and -independent manners. There are at least two mechanisms for BCAS2-mediated AR protein upregulation: One is p53-dependent. The p53 is suppressed by BCAS2 that results in increasing AR mRNA and protein expression. The other is via p53-independent inhibition of proteasome degradation. As BCAS2 can form a complex with AR and HSP90, it may function with HSP90 to stabilise AR protein from being degraded by proteasome. Conclusions: In this study, we show that BCAS2 is a novel AR-interacting protein and characterise the correlation between BCAS2 and PCa. Thus we propose that BCAS2 could be a diagnostic marker and therapeutic target for PCa. PMID:25461807

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

PubMed Central

Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

2010-01-01

Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages

PubMed Central

Miettinen, Juho J.; Matikainen, Sampsa

2012-01-01

Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense. PMID:22973042

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

PubMed Central

2010-01-01

Background Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition. PMID:20704702

High precision multi-genome scale reannotation of enzyme function by EFICAz

PubMed Central

Arakaki, Adrian K; Tian, Weidong; Skolnick, Jeffrey

2006-01-01

Background The functional annotation of most genes in newly sequenced genomes is inferred from similarity to previously characterized sequences, an annotation strategy that often leads to erroneous assignments. We have performed a reannotation of 245 genomes using an updated version of EFICAz, a highly precise method for enzyme function prediction. Results Based on our three-field EC number predictions, we have obtained lower-bound estimates for the average enzyme content in Archaea (29%), Bacteria (30%) and Eukarya (18%). Most annotations added in KEGG from 2005 to 2006 agree with EFICAz predictions made in 2005. The coverage of EFICAz predictions is significantly higher than that of KEGG, especially for eukaryotes. Thousands of our novel predictions correspond to hypothetical proteins. We have identified a subset of 64 hypothetical proteins with low sequence identity to EFICAz training enzymes, whose biochemical functions have been recently characterized and find that in 96% (84%) of the cases we correctly identified their three-field (four-field) EC numbers. For two of the 64 hypothetical proteins: PA1167 from Pseudomonas aeruginosa, an alginate lyase (EC 4.2.2.3) and Rv1700 of Mycobacterium tuberculosis H37Rv, an ADP-ribose diphosphatase (EC 3.6.1.13), we have detected annotation lag of more than two years in databases. Two examples are presented where EFICAz predictions act as hypothesis generators for understanding the functional roles of hypothetical proteins: FLJ11151, a human protein overexpressed in cancer that EFICAz identifies as an endopolyphosphatase (EC 3.6.1.10), and MW0119, a protein of Staphylococcus aureus strain MW2 that we propose as candidate virulence factor based on its EFICAz predicted activity, sphingomyelin phosphodiesterase (EC 3.1.4.12). Conclusion Our results suggest that we have generated enzyme function annotations of high precision and recall. These predictions can be mined and correlated with other information sources to generate biologically significant hypotheses and can be useful for comparative genome analysis and automated metabolic pathway reconstruction. PMID:17166279

Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

PubMed Central

Rose, Annkatrin; Schraegle, Shannon J; Stahlberg, Eric A; Meier, Iris

2005-01-01

Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Results Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. Conclusion MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. PMID:16288662

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

PubMed Central

2014-01-01

Background The chicken eggshell is a natural mechanical barrier to protect egg components from physical damage and microbial penetration. Its integrity and strength is critical for the development of the embryo or to ensure for consumers a table egg free of pathogens. This study compared global gene expression in laying hen uterus in the presence or absence of shell calcification in order to characterize gene products involved in the supply of minerals and / or the shell biomineralization process. Results Microarrays were used to identify a repertoire of 302 over-expressed genes during shell calcification. GO terms enrichment was performed to provide a global interpretation of the functions of the over-expressed genes, and revealed that the most over-represented proteins are related to reproductive functions. Our analysis identified 16 gene products encoding proteins involved in mineral supply, and allowed updating of the general model describing uterine ion transporters during eggshell calcification. A list of 57 proteins potentially secreted into the uterine fluid to be active in the mineralization process was also established. They were classified according to their potential functions (biomineralization, proteoglycans, molecular chaperone, antimicrobials and proteases/antiproteases). Conclusions Our study provides detailed descriptions of genes and corresponding proteins over-expressed when the shell is mineralizing. Some of these proteins involved in the supply of minerals and influencing the shell fabric to protect the egg contents are potentially useful biological markers for the genetic improvement of eggshell quality. PMID:24649854

Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions.

PubMed

Erdozain, Amaia M; De Gois, Stéphanie; Bernard, Véronique; Gorgievski, Victor; Pietrancosta, Nicolas; Dumas, Sylvie; Macedo, Carlos E; Vanhoutte, Peter; Ortega, Jorge E; Meana, J Javier; Tzavara, Eleni T; Vialou, Vincent; Giros, Bruno

2018-04-01

The importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation.

Rapid functional diversification in the structurally conserved ELAV family of neuronal RNA binding proteins

PubMed Central

Samson, Marie-Laure

2008-01-01

Background The Drosophila gene embryonic lethal abnormal visual system (elav) is the prototype of a gene family present in all metazoans. Its members encode structurally conserved neuronal proteins with three RNA Recognition Motifs (RRM) but they paradoxically act at diverse levels of post-transcriptional regulation. In an attempt to understand the history of this family, we searched for orthologs in eleven completely sequenced genomes, including those of humans, D. melanogaster and C. elegans, for which cDNAs are available. Results We analyzed 23 orthologs/paralogs of elav, and found evidence of gain/loss of gene copy number. For one set of genes, including elav itself, the coding sequences are free of introns and their products most resemble ELAV. The remaining genes show remarkable conservation of their exon organization, and their products most resemble FNE and RBP9, proteins encoded by the two elav paralogs of Drosophila. Remarkably, three of the conserved exon junctions are both close to structural elements, involved respectively in protein-RNA interactions and in the regulation of sub-cellular localization, and in the vicinity of diverse sequence variations. Conclusion The data indicate that the essential elav gene of Drosophila is newly emerged, restricted to dipterans and of retrotransposed origin. We propose that the conserved exon junctions constitute potential sites for sequence/function modifications, and that RRM binding proteins, whose function relies upon plastic RNA-protein interactions, may have played an important role in brain evolution. PMID:18715504

Molecular evolution of cyclin proteins in animals and fungi

PubMed Central

2011-01-01

Background The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi. Results We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution. Conclusions The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events. PMID:21798004

Alteration of Cyclic-AMP Response Element Binding Protein in the Postmortem Brain of Subjects with Bipolar Disorder and Schizophrenia

PubMed Central

Ren, Xinguo; Rizavi, Hooriyah S.; Khan, Mansoor A.; Bhaumik, Runa; Dwivedi, Yogesh; Pandey, Ghanshyam N.

2013-01-01

Background Abnormalities of cyclic-AMP (cAMP) response element binding protein (CREB) function has been suggested in bipolar (BP) illness and schizophrenia (SZ), based on both indirect and direct evidence. To further elucidate the role of CREB in these disorders, we studied CREB expression and function in two brain areas implicated in these disorders, i.e., dorsolateral prefrontal cortex (DLPFC) and cingulate gyrus (CG). Methods We determined CREB protein expression using Western blot technique, CRE-DNA binding using gel shift assay, and mRNA expression using real-time RT-polymerase chain reaction (qPCR) in DLPFC and CG of the postmortem brain of BP (n = 19), SZ (n = 20), and normal control (NC, n = 20) subjects. Results We observed that CREB protein and mRNA expression and CRE-DNA binding activity were significantly decreased in the nuclear fraction of DLPFC and CG obtained from BP subjects compared with NC subjects. However, the protein and mRNA expression and CRE-DNA binding in SZ subjects was significantly decreased in CG, but not in DLPFC, compared with NC. Conclusion These studies thus indicate region-specific abnormalities of CREB expression and function in both BP and SZ. They suggest that abnormalities of CREB in CG may be associated with both BP and SZ, but its abnormality in DLPFC is specific to BP illness. PMID:24148789

Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution

PubMed Central

Wolf, Maxim Y; Wolf, Yuri I; Koonin, Eugene V

2008-01-01

Background Proteins show a broad range of evolutionary rates. Understanding the factors that are responsible for the characteristic rate of evolution of a given protein arguably is one of the major goals of evolutionary biology. A long-standing general assumption used to be that the evolution rate is, primarily, determined by the specific functional constraints that affect the given protein. These constrains were traditionally thought to depend both on the specific features of the protein's structure and its biological role. The advent of systems biology brought about new types of data, such as expression level and protein-protein interactions, and unexpectedly, a variety of correlations between protein evolution rate and these variables have been observed. The strongest connections by far were repeatedly seen between protein sequence evolution rate and the expression level of the respective gene. It has been hypothesized that this link is due to the selection for the robustness of the protein structure to mistranslation-induced misfolding that is particularly important for highly expressed proteins and is the dominant determinant of the sequence evolution rate. Results This work is an attempt to assess the relative contributions of protein domain structure and function, on the one hand, and expression level on the other hand, to the rate of sequence evolution. To this end, we performed a genome-wide analysis of the effect of the fusion of a pair of domains in multidomain proteins on the difference in the domain-specific evolutionary rates. The mistranslation-induced misfolding hypothesis would predict that, within multidomain proteins, fused domains, on average, should evolve at substantially closer rates than the same domains in different proteins because, within a mutlidomain protein, all domains are translated at the same rate. We performed a comprehensive comparison of the evolutionary rates of mammalian and plant protein domains that are either joined in multidomain proteins or contained in distinct proteins. Substantial homogenization of evolutionary rates in multidomain proteins was, indeed, observed in both animals and plants, although highly significant differences between domain-specific rates remained. The contributions of the translation rate, as determined by the effect of the fusion of a pair of domains within a multidomain protein, and intrinsic, domain-specific structural-functional constraints appear to be comparable in magnitude. Conclusion Fusion of domains in a multidomain protein results in substantial homogenization of the domain-specific evolutionary rates but significant differences between domain-specific evolution rates remain. Thus, the rate of translation and intrinsic structural-functional constraints both exert sizable and comparable effects on sequence evolution. Reviewers This article was reviewed by Sergei Maslov, Dennis Vitkup, Claus Wilke (nominated by Orly Alter), and Allan Drummond (nominated by Joel Bader). For the full reviews, please go to the Reviewers' Reports section. PMID:18840284

Computational prediction of hinge axes in proteins

PubMed Central

2014-01-01

Background A protein's function is determined by the wide range of motions exhibited by its 3D structure. However, current experimental techniques are not able to reliably provide the level of detail required for elucidating the exact mechanisms of protein motion essential for effective drug screening and design. Computational tools are instrumental in the study of the underlying structure-function relationship. We focus on a special type of proteins called "hinge proteins" which exhibit a motion that can be interpreted as a rotation of one domain relative to another. Results This work proposes a computational approach that uses the geometric structure of a single conformation to predict the feasible motions of the protein and is founded in recent work from rigidity theory, an area of mathematics that studies flexibility properties of general structures. Given a single conformational state, our analysis predicts a relative axis of motion between two specified domains. We analyze a dataset of 19 structures known to exhibit this hinge-like behavior. For 15, the predicted axis is consistent with a motion to a second, known conformation. We present a detailed case study for three proteins whose dynamics have been well-studied in the literature: calmodulin, the LAO binding protein and the Bence-Jones protein. Conclusions Our results show that incorporating rigidity-theoretic analyses can lead to effective computational methods for understanding hinge motions in macromolecules. This initial investigation is the first step towards a new tool for probing the structure-dynamics relationship in proteins. PMID:25080829

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

Application of the principle of linked functions to ATP-driven ion pumps: kinetics of activation by ATP.

PubMed Central

Reynolds, J A; Johnson, E A; Tanford, C

1985-01-01

If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct participant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center. PMID:2987939

Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

PubMed Central

Bartlett, John D.

2013-01-01

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using Library MosSCI

PubMed Central

Kaymak, Ebru; Farley, Brian M.; Hay, Samantha A.; Li, Chihua; Ho, Samantha; Hartman, Daniel J.; Ryder, Sean P.

2016-01-01

Background In C. elegans, germline development and early embryogenesis rely on post-transcriptional regulation of maternally transcribed mRNAs. In many cases, the 3′UTR is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. Results In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RBPs that control the expression pattern of five different maternal mRNAs. Conclusions The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. PMID:27294288

Exaptation of Bornavirus-Like Nucleoprotein Elements in Afrotherians

PubMed Central

Kobayashi, Yuki; Horie, Masayuki; Nakano, Ayumi; Murata, Koichi; Itou, Takuya; Suzuki, Yoshiyuki

2016-01-01

Endogenous bornavirus-like nucleoprotein elements (EBLNs), the nucleotide sequence elements derived from the nucleoprotein gene of ancient bornavirus-like viruses, have been identified in many animal genomes. Here we show evidence that EBLNs encode functional proteins in their host. Some afrotherian EBLNs were observed to have been maintained for more than 83.3 million years under negative selection. Splice variants were expressed from the genomic loci of EBLNs in elephant, and some were translated into proteins. The EBLN proteins appeared to be localized to the rough endoplasmic reticulum in African elephant cells, in contrast to the nuclear localization of bornavirus N. These observations suggest that afrotherian EBLNs have acquired a novel function in their host. Interestingly, genomic sequences of the first exon and its flanking regions in these EBLN loci were homologous to those of transmembrane protein 106B (TMEM106B). The upstream region of the first exon in the EBLN loci exhibited a promoter activity, suggesting that the ability of these EBLNs to be transcribed in the host cell was gained through capturing a partial duplicate of TMEM106B. In conclusion, our results strongly support for exaptation of EBLNs to encode host proteins in afrotherians. PMID:27518265

Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria

PubMed Central

2012-01-01

Background Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. Results By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. Conclusions These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems. PMID:23122209

Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

PubMed Central

2009-01-01

Background Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions. PMID:19889238

Mining protein function from text using term-based support vector machines

PubMed Central

Rice, Simon B; Nenadic, Goran; Stapley, Benjamin J

2005-01-01

Background Text mining has spurred huge interest in the domain of biology. The goal of the BioCreAtIvE exercise was to evaluate the performance of current text mining systems. We participated in Task 2, which addressed assigning Gene Ontology terms to human proteins and selecting relevant evidence from full-text documents. We approached it as a modified form of the document classification task. We used a supervised machine-learning approach (based on support vector machines) to assign protein function and select passages that support the assignments. As classification features, we used a protein's co-occurring terms that were automatically extracted from documents. Results The results evaluated by curators were modest, and quite variable for different problems: in many cases we have relatively good assignment of GO terms to proteins, but the selected supporting text was typically non-relevant (precision spanning from 3% to 50%). The method appears to work best when a substantial set of relevant documents is obtained, while it works poorly on single documents and/or short passages. The initial results suggest that our approach can also mine annotations from text even when an explicit statement relating a protein to a GO term is absent. Conclusion A machine learning approach to mining protein function predictions from text can yield good performance only if sufficient training data is available, and significant amount of supporting data is used for prediction. The most promising results are for combined document retrieval and GO term assignment, which calls for the integration of methods developed in BioCreAtIvE Task 1 and Task 2. PMID:15960835

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement.

PubMed

Caswell, Clayton C; Han, Runlin; Hovis, Kelley M; Ciborowski, Pawel; Keene, Douglas R; Marconi, Richard T; Lukomski, Slawomir

2008-02-01

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

Alternative ground states enable pathway switching in biological electron transfer

DOE PAGES

Abriata, Luciano A.; Alvarez-Paggi, Damian; Ledesma, Gabirela N.; ...

2012-10-10

Electron transfer is the simplest chemical reaction and constitutes the basis of a large variety of biological processes, such as photosynthesis and cellular respiration. Nature has evolved specific proteins and cofactors for these functions. The mechanisms optimizing biological electron transfer have been matter of intense debate, such as the role of the protein milieu between donor and acceptor sites. Here we propose a mechanism regulating long-range electron transfer in proteins. Specifically, we report a spectroscopic, electrochemical, and theoretical study on WT and single-mutant CuA redox centers from Thermus thermophilus, which shows that thermal fluctuations may populate two alternative ground-state electronicmore » wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways. In conclusion, these findings suggest a unique role for alternative or “invisible” electronic ground states in directional electron transfer. Moreover, it is shown that this energy gap and, therefore, the equilibrium between ground states can be fine-tuned by minor perturbations, suggesting alternative ways through which protein–protein interactions and membrane potential may optimize and regulate electron–proton energy transduction.« less

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

Soluble expression, purification and characterization of the full length IS2 Transposase

PubMed Central

2011-01-01

Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Conclusions Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level. PMID:22032517

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

PubMed Central

Maroto, Rosario; Zhao, Yingxin; Jamaluddin, Mohammad; Popov, Vsevolod L.; Wang, Hongwang; Kalubowilage, Madumali; Zhang, Yueqing; Luisi, Jonathan; Sun, Hong; Culbertson, Christopher T.; Bossmann, Stefan H.; Motamedi, Massoud; Brasier, Allan R.

2017-01-01

ABSTRACT Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation. PMID:28819550

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses.

PubMed

Maroto, Rosario; Zhao, Yingxin; Jamaluddin, Mohammad; Popov, Vsevolod L; Wang, Hongwang; Kalubowilage, Madumali; Zhang, Yueqing; Luisi, Jonathan; Sun, Hong; Culbertson, Christopher T; Bossmann, Stefan H; Motamedi, Massoud; Brasier, Allan R

2017-01-01

Background : Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim : We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods : Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results : Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions : Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Pfarao: a web application for protein family analysis customized for cytoskeletal and motor proteins (CyMoBase)

PubMed Central

Odronitz, Florian; Kollmar, Martin

2006-01-01

Background Annotation of protein sequences of eukaryotic organisms is crucial for the understanding of their function in the cell. Manual annotation is still by far the most accurate way to correctly predict genes. The classification of protein sequences, their phylogenetic relation and the assignment of function involves information from various sources. This often leads to a collection of heterogeneous data, which is hard to track. Cytoskeletal and motor proteins consist of large and diverse superfamilies comprising up to several dozen members per organism. Up to date there is no integrated tool available to assist in the manual large-scale comparative genomic analysis of protein families. Description Pfarao (Protein Family Application for Retrieval, Analysis and Organisation) is a database driven online working environment for the analysis of manually annotated protein sequences and their relationship. Currently, the system can store and interrelate a wide range of information about protein sequences, species, phylogenetic relations and sequencing projects as well as links to literature and domain predictions. Sequences can be imported from multiple sequence alignments that are generated during the annotation process. A web interface allows to conveniently browse the database and to compile tabular and graphical summaries of its content. Conclusion We implemented a protein sequence-centric web application to store, organize, interrelate, and present heterogeneous data that is generated in manual genome annotation and comparative genomics. The application has been developed for the analysis of cytoskeletal and motor proteins (CyMoBase) but can easily be adapted for any protein. PMID:17134497

Four signature motifs define the first class of structurally related large coiled-coil proteins in plants.

PubMed Central

Gindullis, Frank; Rose, Annkatrin; Patel, Shalaka; Meier, Iris

2002-01-01

Background Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants. Results We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP) were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms. Conclusion Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom. PMID:11972898

Metaproteomics Provides Functional Insight into Activated Sludge Wastewater Treatment

PubMed Central

Wilmes, Paul; Wexler, Margaret; Bond, Philip L.

2008-01-01

Background Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR). Methodology/Principal Findings A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. Conclusions/Significance Importantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models. PMID:18392150

A role for the membrane protein M6 in the Drosophila visual system

PubMed Central

2012-01-01

Background Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system. Results The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light. Conclusions Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals. PMID:22762289

Comparison of Renal Function and Other Predictors in Lacto-Ovo Vegetarians and Omnivores With Chronic Kidney Disease.

PubMed

Chang, Chou-Yueh; Chang, Horng-Rong; Lin, Hsing-Chun; Chang, Han-Hsin

2018-03-13

Objective Vegetarian diets have been shown to increase the risk of certain nutritional deficiencies, such as iron. As a number of patients with chronic kidney disease (CKD) in Taiwan are lacto-ovo vegetarians, the aim of this study was to investigate the effects of different proportions and sources of protein in lacto-ovo vegetarian and omnivorous diets, as well as the influence of adequate dietary protein intake, on renal function and nutritional status of Taiwanese patients with stage 3 to stage 5 CKD. Methods This is a cross-sectional study. In total, 100 outpatients with stage 3 to stage 5 CKD were enrolled in this study, including 40 lacto-ovo vegetarians and 60 omnivores. Subjects were divided into the lacto-ovo vegetarian group and omnivorous group based on dietary protein patterns. The indicators of renal function included estimated glomerular filtration rate (eGFR), creatinine, and blood urea nitrogen (BUN). Albumin, hemoglobin (Hb), and red blood cell count (RBC) measurements served as nutritional indicators. The levels of dietary energy and protein, as well as protein sources (plant or animal), were also analyzed. Results The levels of serum phosphate and triglycerides were significantly lower in the lacto-ovo vegetarian group than in the omnivore group, suggesting that lacto-ovo vegetarian diets have both phosphate-lowering and lipid-lowering effects, which could reduce the development of hyperphosphatemia and dyslipidemia. However, since all groups consumed higher than the recommended amounts of protein diet intake, no significant differences were observed in other renal function indices between the two groups. Conclusion Although a larger cohort study is necessary, the findings of this study could help patients with CKD to make healthier food choices and be used to support future medical nutritional therapies.

Assessment of the potential pathogenicity of missense mutations identified in the GTPase-activating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene.

PubMed

Thomas, Laura; Richards, Mark; Mort, Matthew; Dunlop, Elaine; Cooper, David N; Upadhyaya, Meena

2012-12-01

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function. © 2012 Wiley Periodicals, Inc.

From cyanobacteria to plants: conservation of PII functions during plastid evolution.

PubMed

Chellamuthu, Vasuki Ranjani; Alva, Vikram; Forchhammer, Karl

2013-02-01

This article reviews the current state-of-the-art concerning the functions of the signal processing protein PII in cyanobacteria and plants, with a special focus on evolutionary aspects. We start out with a general introduction to PII proteins, their distribution, and their evolution. We also discuss PII-like proteins and domains, in particular, the similarity between ATP-phosphoribosyltransferase (ATP-PRT) and its PII-like domain and the complex between N-acetyl-L-glutamate kinase (NAGK) and its PII activator protein from oxygenic phototrophs. The structural basis of the function of PII as an ATP/ADP/2-oxoglutarate signal processor is described for Synechococcus elongatus PII. In both cyanobacteria and plants, a major target of PII regulation is NAGK, which catalyzes the committed step of arginine biosynthesis. The common principles of NAGK regulation by PII are outlined. Based on the observation that PII proteins from cyanobacteria and plants can functionally replace each other, the hypothesis that PII-dependent NAGK control was under selective pressure during the evolution of plastids of Chloroplastida and Rhodophyta is tested by bioinformatics approaches. It is noteworthy that two lineages of heterokont algae, diatoms and brown algae, also possess NAGK, albeit lacking PII; their NAGK however appears to have descended from an alphaproteobacterium and not from a cyanobacterium as in plants. We end this article by coming to the conclusion that during the evolution of plastids, PII lost its function in coordinating gene expression through the PipX-NtcA network but preserved its role in nitrogen (arginine) storage metabolism, and subsequently took over the fine-tuned regulation of carbon (fatty acid) storage metabolism, which is important in certain developmental stages of plants.

COPI selectively drives maturation of the early Golgi

PubMed Central

Papanikou, Effrosyni; Day, Kasey J; Austin, Jotham; Glick, Benjamin S

2015-01-01

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins. DOI: http://dx.doi.org/10.7554/eLife.13232.001 PMID:26709839

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

PubMed Central

2012-01-01

Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain annotation by identifying those pattern residues that most distinguish each protein domain subgroup from other related subgroups. PMID:22726767

WS-SNPs&GO: a web server for predicting the deleterious effect of human protein variants using functional annotation

PubMed Central

2013-01-01

Background SNPs&GO is a method for the prediction of deleterious Single Amino acid Polymorphisms (SAPs) using protein functional annotation. In this work, we present the web server implementation of SNPs&GO (WS-SNPs&GO). The server is based on Support Vector Machines (SVM) and for a given protein, its input comprises: the sequence and/or its three-dimensional structure (when available), a set of target variations and its functional Gene Ontology (GO) terms. The output of the server provides, for each protein variation, the probabilities to be associated to human diseases. Results The server consists of two main components, including updated versions of the sequence-based SNPs&GO (recently scored as one of the best algorithms for predicting deleterious SAPs) and of the structure-based SNPs&GO3d programs. Sequence and structure based algorithms are extensively tested on a large set of annotated variations extracted from the SwissVar database. Selecting a balanced dataset with more than 38,000 SAPs, the sequence-based approach achieves 81% overall accuracy, 0.61 correlation coefficient and an Area Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve of 0.88. For the subset of ~6,600 variations mapped on protein structures available at the Protein Data Bank (PDB), the structure-based method scores with 84% overall accuracy, 0.68 correlation coefficient, and 0.91 AUC. When tested on a new blind set of variations, the results of the server are 79% and 83% overall accuracy for the sequence-based and structure-based inputs, respectively. Conclusions WS-SNPs&GO is a valuable tool that includes in a unique framework information derived from protein sequence, structure, evolutionary profile, and protein function. WS-SNPs&GO is freely available at http://snps.biofold.org/snps-and-go. PMID:23819482

ΔΔPT: a comprehensive toolbox for the analysis of protein motion

PubMed Central

2013-01-01

Background Normal Mode Analysis is one of the most successful techniques for studying motions in proteins and macromolecules. It can provide information on the mechanism of protein functions, used to aid crystallography and NMR data reconstruction, and calculate protein free energies. Results ΔΔPT is a toolbox allowing calculation of elastic network models and principle component analysis. It allows the analysis of pdb files or trajectories taken from; Gromacs, Amber, and DL_POLY. As well as calculation of the normal modes it also allows comparison of the modes with experimental protein motion, variation of modes with mutation or ligand binding, and calculation of molecular dynamic entropies. Conclusions This toolbox makes the respective tools available to a wide community of potential NMA users, and allows them unrivalled ability to analyse normal modes using a variety of techniques and current software. PMID:23758746

Poliovirus 2C protein forms homo-oligomeric structures required for ATPase activity.

PubMed

Adams, Peter; Kandiah, Eaazhisai; Effantin, Grégory; Steven, Alasdair C; Ehrenfeld, Ellie

2009-08-14

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.

Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity*

PubMed Central

Adams, Peter; Kandiah, Eaazhisai; Effantin, Grégory; Steven, Alasdair C.; Ehrenfeld, Ellie

2009-01-01

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5–8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain. PMID:19520852

[Relationships between venomous function and innate immune function].

PubMed

Goyffon, Max; Saul, Frederick; Faure, Grazyna

2015-01-01

Venomous function is investigated in relation to innate immune function in two cases selected from scorpion venom and serpent venom. In the first case, structural analysis of scorpion toxins and defensins reveals a close interrelation between both functions (toxic and innate immune system function). In the second case, structural and functional studies of natural inhibitors of toxic snake venom phospholipases A2 reveal homology with components of the innate immune system, leading to a similar conclusion. Although there is a clear functional distinction between neurotoxins, which act by targeting membrane ion channels, and the circulating defensins which protect the organism from pathogens, the scorpion short toxins and defensins share a common protein folding scaffold with a conserved cysteine-stabilized alpha-beta motif of three disulfide bridges linking a short alpha helix and an antiparallel beta sheet. Genomic analysis suggests that these proteins share a common ancestor (long venom toxins were separated from an early gene family which gave rise to separate short toxin and defensin families). Furthermore, a scorpion toxin has been experimentally synthetized from an insect defensin, and an antibacterial scorpion peptide, androctonin (whose structure is similar to that of a cone snail venom toxin), was shown to have a similar high affinity for the postsynaptic acetylcholine receptor of Torpedo sp. Natural inhibitors of phospholipase A2 found in the blood of snakes are associated with the resistance of venomous snakes to their own highly neurotoxic venom proteins. Three classes of phospholipases A2 inhibitors (PLI-α, PLI-β, PLI-γ) have been identified. These inhibitors display diverse structural motifs related to innate immune proteins including carbohydrate recognition domains (CRD), leucine rich repeat domains (found in Toll-like receptors) and three finger domains, which clearly differentiate them from components of the adaptive immune system. Thus, in structure, function and phylogeny, venomous function in both vertebrates and invertebrates are clearly interrelated with innate immune function. © Société de Biologie, 2016.

Low-frequency transcranial magnetic stimulation is beneficial for enhancing synaptic plasticity in the aging brain.

PubMed

Zhang, Zhan-Chi; Luan, Feng; Xie, Chun-Yan; Geng, Dan-Dan; Wang, Yan-Yong; Ma, Jun

2015-06-01

In the aging brain, cognitive function gradually declines and causes a progressive reduction in the structural and functional plasticity of the hippocampus. Transcranial magnetic stimulation is an emerging and novel neurological and psychiatric tool used to investigate the neurobiology of cognitive function. Recent studies have demonstrated that low-frequency transcranial magnetic stimulation (≤1 Hz) ameliorates synaptic plasticity and spatial cognitive deficits in learning-impaired mice. However, the mechanisms by which this treatment improves these deficits during normal aging are still unknown. Therefore, the current study investigated the effects of transcranial magnetic stimulation on the brain-derived neurotrophic factor signal pathway, synaptic protein markers, and spatial memory behavior in the hippocampus of normal aged mice. The study also investigated the downstream regulator, Fyn kinase, and the downstream effectors, synaptophysin and growth-associated protein 43 (both synaptic markers), to determine the possible mechanisms by which transcranial magnetic stimulation regulates cognitive capacity. Transcranial magnetic stimulation with low intensity (110% average resting motor threshold intensity, 1 Hz) increased mRNA and protein levels of brain-derived neurotrophic factor, tropomyosin receptor kinase B, and Fyn in the hippocampus of aged mice. The treatment also upregulated the mRNA and protein expression of synaptophysin and growth-associated protein 43 in the hippocampus of these mice. In conclusion, brain-derived neurotrophic factor signaling may play an important role in sustaining and regulating structural synaptic plasticity induced by transcranial magnetic stimulation in the hippocampus of aging mice, and Fyn may be critical during this regulation. These responses may change the structural plasticity of the aging hippocampus, thereby improving cognitive function.

HMMER Cut-off Threshold Tool (HMMERCTTER): Supervised classification of superfamily protein sequences with a reliable cut-off threshold

PubMed Central

Pagnuco, Inti Anabela; Revuelta, María Victoria; Bondino, Hernán Gabriel; Brun, Marcel

2018-01-01

Background Protein superfamilies can be divided into subfamilies of proteins with different functional characteristics. Their sequences can be classified hierarchically, which is part of sequence function assignation. Typically, there are no clear subfamily hallmarks that would allow pattern-based function assignation by which this task is mostly achieved based on the similarity principle. This is hampered by the lack of a score cut-off that is both sensitive and specific. Results HMMER Cut-off Threshold Tool (HMMERCTTER) adds a reliable cut-off threshold to the popular HMMER. Using a high quality superfamily phylogeny, it clusters a set of training sequences such that the cluster-specific HMMER profiles show cluster or subfamily member detection with 100% precision and recall (P&R), thereby generating a specific threshold as inclusion cut-off. Profiles and thresholds are then used as classifiers to screen a target dataset. Iterative inclusion of novel sequences to groups and the corresponding HMMER profiles results in high sensitivity while specificity is maintained by imposing 100% P&R self detection. In three presented case studies of protein superfamilies, classification of large datasets with 100% precision was achieved with over 95% recall. Limits and caveats are presented and explained. Conclusions HMMERCTTER is a promising protein superfamily sequence classifier provided high quality training datasets are used. It provides a decision support system that aids in the difficult task of sequence function assignation in the twilight zone of sequence similarity. All relevant data and source codes are available from the Github repository at the following URL: https://github.com/BBCMdP/HMMERCTTER. PMID:29579071

Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34.

PubMed

Ott, Melanie; Tascher, Georg; Hassdenteufel, Sarah; Zimmermann, Richard; Haas, Jürgen; Bailer, Susanne M

2011-12-01

Release of herpes simplex virus type 1 (HSV-1) nucleocapsids from the host nucleus relies on the nuclear egress complex consisting of the two essential proteins pUL34 and pUL31. The cytoplasmically exposed N-terminal region of pUL34 interacts with pUL31, while a hydrophobic region followed by a short luminal part mediates membrane association. Based on its domain organization, pUL34 was postulated to be a tail-anchor (TA) protein. We performed a coupled in vitro transcription/translation assay to show that membrane insertion of pUL34 occurs post-translationally. Transient transfection and localization experiments in mammalian cells were combined with HSV-1 bacterial artificial chromosome mutagenesis to reveal the functional properties of the essential pUL34 TA. Our data show that a minimal tail length of 15 residues is sufficient for nuclear envelope targeting and pUL34 function. Permutations of the pUL34 TA with orthologous regions of human cytomegalovirus pUL50 or Epstein-Barr virus pBFRF1 as well as the heterologous HSV-1 TA proteins pUL56 or pUS9 or the cellular TA proteins Bcl-2 and Vamp2 revealed that nuclear egress tolerates TAs varying in sequence and hydrophobicity, while a non-α-helical membrane anchor failed to complement the pUL34 function. In conclusion, this study provides the first mechanistic insights into the particular role of the TA of pUL34 in membrane curving and capsid egress from the host nucleus.

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

PubMed Central

Kurscheid, Sebastian; Lew-Tabor, Ala E; Rodriguez Valle, Manuel; Bruyeres, Anthea G; Doogan, Vivienne J; Munderloh, Ulrike G; Guerrero, Felix D; Barrero, Roberto A; Bellgard, Matthew I

2009-01-01

Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects. PMID:19323841

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

PubMed

Wei, Dong; Tian, Chuan-Bei; Liu, Shi-Huo; Wang, Tao; Smagghe, Guy; Jia, Fu-Xian; Dou, Wei; Wang, Jin-Jun

2016-06-01

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest. Copyright © 2015 Elsevier Inc. All rights reserved.

UBIAD1 Mutation Alters a Mitochondrial Prenyltransferase to Cause Schnyder Corneal Dystrophy

PubMed Central

Nickerson, Michael L.; Kostiha, Brittany N.; Brandt, Wolfgang; Fredericks, William; Xu, Ke-Ping; Yu, Fu-Shin; Gold, Bert; Chodosh, James; Goldberg, Marc; Lu, Da Wen; Yamada, Masakazu; Tervo, Timo M.; Grutzmacher, Richard; Croasdale, Chris; Hoeltzenbein, Maria; Sutphin, John; Malkowicz, S. Bruce; Wessjohann, Ludger; Kruth, Howard S.; Dean, Michael; Weiss, Jayne S.

2010-01-01

Background Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure. Methodology/Principal Findings We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules. Conclusions/Significance Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner. PMID:20505825

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

PubMed Central

Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

2016-01-01

Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients. PMID:27104530

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

PubMed

Antoniassi, Mariana Pereira; Intasqui, Paula; Camargo, Mariana; Zylbersztejn, Daniel Suslik; Carvalho, Valdemir Melechco; Cardozo, Karina H M; Bertolla, Ricardo Pimenta

2016-11-01

To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

Unfoldomics of human diseases: linking protein intrinsic disorder with diseases

PubMed Central

Uversky, Vladimir N; Oldfield, Christopher J; Midic, Uros; Xie, Hongbo; Xue, Bin; Vucetic, Slobodan; Iakoucheva, Lilia M; Obradovic, Zoran; Dunker, A Keith

2009-01-01

Background Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack stable tertiary and/or secondary structure yet fulfills key biological functions. The recent recognition of IDPs and IDRs is leading to an entire field aimed at their systematic structural characterization and at determination of their mechanisms of action. Bioinformatics studies showed that IDPs and IDRs are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition and signal transduction. These activities complement the functions of structured proteins. IDPs and IDRs were shown to participate in both one-to-many and many-to-one signaling. Alternative splicing and posttranslational modifications are frequently used to tune the IDP functionality. Several individual IDPs were shown to be associated with human diseases, such as cancer, cardiovascular disease, amyloidoses, diabetes, neurodegenerative diseases, and others. This raises questions regarding the involvement of IDPs and IDRs in various diseases. Results IDPs and IDRs were shown to be highly abundant in proteins associated with various human maladies. As the number of IDPs related to various diseases was found to be very large, the concepts of the disease-related unfoldome and unfoldomics were introduced. Novel bioinformatics tools were proposed to populate and characterize the disease-associated unfoldome. Structural characterization of the members of the disease-related unfoldome requires specialized experimental approaches. IDPs possess a number of unique structural and functional features that determine their broad involvement into the pathogenesis of various diseases. Conclusion Proteins associated with various human diseases are enriched in intrinsic disorder. These disease-associated IDPs and IDRs are real, abundant, diversified, vital, and dynamic. These proteins and regions comprise the disease-related unfoldome, which covers a significant part of the human proteome. Profound association between intrinsic disorder and various human diseases is determined by a set of unique structural and functional characteristics of IDPs and IDRs. Unfoldomics of human diseases utilizes unrivaled bioinformatics and experimental techniques, paves the road for better understanding of human diseases, their pathogenesis and molecular mechanisms, and helps develop new strategies for the analysis of disease-related proteins. PMID:19594884

Long-Term Effects of a Randomised Controlled Trial Comparing High Protein or High Carbohydrate Weight Loss Diets on Testosterone, SHBG, Erectile and Urinary Function in Overweight and Obese Men

PubMed Central

Moran, Lisa J.; Brinkworth, Grant D.; Martin, Sean; Wycherley, Thomas P.; Stuckey, Bronwyn; Lutze, Janna; Clifton, Peter M.; Wittert, Gary A.; Noakes, Manny

2016-01-01

Introduction Obesity is associated with reduced testosterone and worsened erectile and sexual function in men. Weight loss improves these outcomes. High protein diets potentially offer anthropometric and metabolic benefits, but their effects on reproductive and sexual outcomes is not known. Aim To examine the long-term effects of weight loss with a higher protein or carbohydrate diet on testosterone, sex hormone binding globulin, erectile dysfunction, lower urinary tract symptoms and sexual desire in overweight and obese men. Methods One-hundred and eighteen overweight or obese men (body mass index 27–40 kg/m2, age 20–65 years) were randomly assigned to an energy restricted higher protein low fat (35% protein, 40% carbohydrate, 25% fat; n = 57) or higher carbohydrate low fat diet (17% protein, 58% carbohydrate, 25% fat, n = 61) diet for 52 weeks (12 weeks weight loss, 40 weeks weight maintenance). Primary outcomes were serum total testosterone, sex hormone binding globulin and calculated free testosterone. Secondary outcomes were erectile function as assessed by the International Index of Erectile Function (IIEF) (total score and erectile function domain), lower urinary tract symptoms and sexual desire. Results Total testosterone, sex hormone binding globulin and free testosterone increased (P<0.001) and the total IIEF increased (P = 0.017) with no differences between diets (P≥0.244). Increases in testosterone (P = 0.037) and sex hormone binding globulin (P<0.001) and improvements in the total IIEF (P = 0.041) occurred from weeks 0–12 with a further increase in testosterone from week 12–52 (P = 0.002). Increases in free testosterone occurred from week 12–52 (p = 0.002). The IIEF erectile functon domain, lower urinary tract symptoms and sexual desire did not change in either group (P≥0.126). Conclusions In overweight and obese men, weight loss with both high protein and carbohydrate diets improve testosterone, sex hormone binding globulin and overall sexual function. Trial Registration Anzctr.org.au ACTRN12606000002583 PMID:27584019

Receptor Tyrosine Kinase MET Interactome and Neurodevelopmental Disorder Partners at the Developing Synapse

PubMed Central

Xie, Zhihui; Li, Jing; Baker, Jonathan; Eagleson, Kathie L.; Coba, Marcelo P.; Levitt, Pat

2016-01-01

Background Atypical synapse development and plasticity are implicated in many neurodevelopmental disorders (NDDs). NDD-associated, high confidence risk genes have been identified, yet little is known about functional relationships at the level of protein-protein interactions, which are the dominant molecular bases responsible for mediating circuit development. Methods Proteomics in three independent developing neocortical synaptosomal preparations identified putative interacting proteins of the ligand-activated MET receptor tyrosine kinase, an autism risk gene that mediates synapse development. The candidates were translated into interactome networks and analyzed bioinformatically. Additionally, three independent quantitative proximity ligation assays (PLA) in cultured neurons and four independent immunoprecipitation analyses of synaptosomes validated protein interactions. Results Approximately 11% (8/72) of MET-interacting proteins, including SHANK3, SYNGAP1 and GRIN2B, are associated with NDDs. Proteins in the MET interactome were translated into a novel MET interactome network based on human protein-protein interaction databases. High confidence genes from different NDD datasets that encode synaptosomal proteins were analyzed for being enriched in MET interactome proteins. This was found for autism, but not schizophrenia, bipolar disorder, major depressive disorder or attentional deficit hyperactivity disorder. There is correlated gene expression between MET and its interactive partners in developing human temporal and visual neocortices, but not with highly expressed genes that are not in the interactome. PLA and biochemical analyses demonstrate that MET-protein partner interactions are dynamically regulated by receptor activation. Conclusions The results provide a novel molecular framework for deciphering the functional relations of key regulators of synaptogenesis that contribute to both typical cortical development and to NDDs. PMID:27086544

A longitudinal analysis of circulating stress-related proteins and chronic ethanol self-administration in cynomolgus macaques

PubMed Central

Helms, Christa M.; Messaoudi, Ilhem; Jeng, Sophia; Freeman, Willard M.; Vrana, Kent E.; Grant, Kathleen A.

2011-01-01

Background Alcoholics have alterations in endocrine and immune function and increased susceptibility to stress-related disorders. A longitudinal analysis of chronic ethanol intake on homeostatic mechanisms is, however, incompletely characterized in primates. Methods Plasma proteins (n = 60; Luminex) and hormones (adrenocorticotropic hormone, ACTH; cortisol) were repeatedly measured in adult male cynomolgus monkeys (Macaca fascicularis, n = 10) during a 32-month experimental protocol at baseline, during induction of water and ethanol (4% w/v in water) self-administration, after 4 months and after 12 months of 22-h daily concurrent access to ethanol and water. Results Significant changes were observed in ACTH, cortisol and 45/60 plasma proteins: a majority (28/45) were suppressed as a function of ethanol self-administration, eight proteins were elevated and nine showed biphasic changes. Cortisol and ACTH were greatest during induction, and correlations between these hormones and plasma proteins varied across the experiment. Pathway analyses implicated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) as possible mediators of ethanol-induced effects on immune-related proteins in primates. Conclusion Chronic ethanol consumption in primates leads to an allostatic state of physiological compromise with respect to circulating immune- and stress-related proteins in NF-κB- and STAT/JAK-related pathways in correlation with altered endocrine activity. PMID:22141444

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

PubMed Central

2011-01-01

Background Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme. Results We found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nme's function in migratory processes was engaged long before the composition of true tissues. Conclusions This study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. PMID:21457554

New statistical potential for quality assessment of protein models and a survey of energy functions

PubMed Central

2010-01-01

Background Scoring functions, such as molecular mechanic forcefields and statistical potentials are fundamentally important tools in protein structure modeling and quality assessment. Results The performances of a number of publicly available scoring functions are compared with a statistical rigor, with an emphasis on knowledge-based potentials. We explored the effect on accuracy of alternative choices for representing interaction center types and other features of scoring functions, such as using information on solvent accessibility, on torsion angles, accounting for secondary structure preferences and side chain orientation. Partially based on the observations made, we present a novel residue based statistical potential, which employs a shuffled reference state definition and takes into account the mutual orientation of residue side chains. Atom- and residue-level statistical potentials and Linux executables to calculate the energy of a given protein proposed in this work can be downloaded from http://www.fiserlab.org/potentials. Conclusions Among the most influential terms we observed a critical role of a proper reference state definition and the benefits of including information about the microenvironment of interaction centers. Molecular mechanical potentials were also tested and found to be over-sensitive to small local imperfections in a structure, requiring unfeasible long energy relaxation before energy scores started to correlate with model quality. PMID:20226048

Conformational landscapes of membrane proteins delineated by enhanced sampling molecular dynamics simulations.

PubMed

Harpole, Tyler J; Delemotte, Lucie

2018-04-01

The expansion of computational power, better parameterization of force fields, and the development of novel algorithms to enhance the sampling of the free energy landscapes of proteins have allowed molecular dynamics (MD) simulations to become an indispensable tool to understand the function of biomolecules. The temporal and spatial resolution of MD simulations allows for the study of a vast number of processes of interest. Here, we review the computational efforts to uncover the conformational free energy landscapes of a subset of membrane proteins: ion channels, transporters and G-protein coupled receptors. We focus on the various enhanced sampling techniques used to study these questions, how the conclusions come together to build a coherent picture, and the relationship between simulation outcomes and experimental observables. Copyright © 2017 Elsevier B.V. All rights reserved.

Dipeptidyl peptidase (DP) 6 and DP10: novel brain proteins implicated in human health and disease.

PubMed

McNicholas, Kym; Chen, Tong; Abbott, Catherine A

2009-01-01

Dipeptidyl peptidase (DP) 6 and DP10 are non-enzyme members of the dipeptidyl peptidase IV family, which includes fibroblast activation protein, DP8, and DP9. DP6 and DP10 proteins have been shown to be critical components of voltage-gated potassium (Kv) channels important in determining cellular excitability. The aim of this paper was to review the research to date on DP6 and DP10 structure, expression, and functions. To date, the protein region responsible for modulating Kv4 channels has not been conclusively identified and the significance of the splice variants has not been resolved. Resolution of these issues will improve our overall knowledge of DP6 and DP10 and lead to a better understanding of their role in diseases, such as asthma and Alzheimer's disease.

The gene transformer-2 of Sciara (Diptera, Nematocera) and its effect on Drosophila sexual development

PubMed Central

2011-01-01

Background The gene transformer-2, which is involved in sex determination, has been studied in Drosophila, Musca, Ceratitis, Anastrepha and Lucilia. All these members of Diptera belong to the suborder Brachycera. In this work, it is reported the isolation and characterisation of genes transformer-2 of the dipterans Sciara ocellaris and Bradysia coprophila (formerly Sciara coprophila), which belong to the much less extensively analysed Sciaridae Family of the Suborder Nematocera, which is paraphyletic with respect to Suborder Brachycera. Results The transformer-2 genes of the studied Sciara species were found to be transcribed in both sexes during development and adult life, in both the soma and germ lines. They produced a single primary transcript, which follows the same alternative splicing in both sexes, giving rise to different mRNAs isoforms. In S. ocellaris the most abundant mRNA isoform encoded a full-length protein of 251 amino acids, while that of B. coprophila encoded a protein of 246 amino acids. Both showed the features of the SR protein family. The less significant mRNA isoforms of both species encoded truncated, presumably non-functional Transformer-2 proteins. The comparison of the functional Sciara Transformer-2 proteins among themselves and those of other insects revealed the greatest degree of conservation in the RRM domain and linker region. In contrast, the RS1 and RS2 domains showed extensive variation with respect to their number of amino acids and their arginine-serine (RS) dipeptide content. The expression of S. ocellaris Transformer-2 protein in Drosophila XX pseudomales lacking the endogenous transformer-2 function caused their partial feminisation. Conclusions The transformer-2 genes of both Sciaridae species encode a single protein in both sexes that shares the characteristics of the Transformer-2 proteins of other insects. These proteins showed conserved sex-determination function in Drosophila; i.e., they were able to form a complex with the endogenous Drosophila Transformer protein that controls the female-specific splicing of the Drosophila doublesex pre-mRNA. However, it appears that the complex formed between the Drosophila Transformer protein and the Sciara Transformer-2 protein is less effective at inducing the female-specific splicing of the endogenous Drosophila doublesex pre-mRNA than the DrosophilaTransformer-Transformer2 complex. This suggests the existence of species-specific co-evolution of the Transformer and Transformer-2 proteins. PMID:21406087

Quality assessment of protein model-structures based on structural and functional similarities

PubMed Central

2012-01-01

Background Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. Results GOBA - Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. Conclusions The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and one of CASP9, compared to the contest participants. Consequently, GOBA offers a novel single model quality assessment program that addresses the practical needs of biologists. In conjunction with other Model Quality Assessment Programs (MQAPs), it would prove useful for the evaluation of single protein models. PMID:22998498

The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains

PubMed Central

Ren, Siyuan; Yang, Guang; He, Youyu; Wang, Yiguo; Li, Yixue; Chen, Zhengjun

2008-01-01

Background Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often < 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Results Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. Conclusion The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains. PMID:18828911

Effect of aquatic exercise on mental health, functional autonomy, and oxidative dysfunction in hypertensive adults.

PubMed

Da Silva, Luciano Acordi; Menguer, Lorhan; Motta, Janaina; Dieke, Beatriz; Mariano, Sindianra; Tasca, Gladson; Zacaron, Rubya Pereira; Silveira, Paulo Cesar Lock; Aurino, Pinho Ricardo

2017-11-27

The aquatic exercise is an effective non-pharmacological therapy for prevention and control of hypertension. The objective of the present study was to investigate the effect of aquatic exercise on mental health, functional autonomy, and oxidative dysfunction in hypertensive adults. Methodologically 29 adults (mean age 53 ± 7.5 years) were included in the study, and were randomly grouped as hypertensive (n = 16) and non-hypertensive (n = 13). Both groups underwent low-intensity aquatic exercise program for 12 weeks. Outcomes were evaluated at week 0 and 12. The values for the following parameters decreased in the hypertensive group post training: anxiety (-6.2 ± 2 score; 60%), Timed Up and Go test (-7.4 ± 0.3 sec; 30%), protein carbonylation (-0.15 ± 0.03 nmol/mg protein; 50%), nitric oxide (12.4 ± 6 nmol/mg protein; 62%), interleukin-6 (-27.6 ± 5.7 pg/mg protein; 46%), and tissue necrosis factor-alpha (-52.4 ± 3.8 pg/mg protein; 40%); however, the values of the following parameters increased before training: Berg score (56 ± 2; 7.8%), flexibility (27 ± 1 cm; 71%); glutathione (3.1 ± 1.3 nmol/mg protein; 138%), and superoxide dismutase (1.6 ± 0.4 nmol/mg; 166%). In conclusion, we suggest that low-intensity aquatic exercise program improved anxiety, functional autonomy, and oxidative dysfunction in hypertensive adults.

Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae

PubMed Central

Reguly, Teresa; Breitkreutz, Ashton; Boucher, Lorrie; Breitkreutz, Bobby-Joe; Hon, Gary C; Myers, Chad L; Parsons, Ainslie; Friesen, Helena; Oughtred, Rose; Tong, Amy; Stark, Chris; Ho, Yuen; Botstein, David; Andrews, Brenda; Boone, Charles; Troyanskya, Olga G; Ideker, Trey; Dolinski, Kara; Batada, Nizar N; Tyers, Mike

2006-01-01

Background The study of complex biological networks and prediction of gene function has been enabled by high-throughput (HTP) methods for detection of genetic and protein interactions. Sparse coverage in HTP datasets may, however, distort network properties and confound predictions. Although a vast number of well substantiated interactions are recorded in the scientific literature, these data have not yet been distilled into networks that enable system-level inference. Results We describe here a comprehensive database of genetic and protein interactions, and associated experimental evidence, for the budding yeast Saccharomyces cerevisiae, as manually curated from over 31,793 abstracts and online publications. This literature-curated (LC) dataset contains 33,311 interactions, on the order of all extant HTP datasets combined. Surprisingly, HTP protein-interaction datasets currently achieve only around 14% coverage of the interactions in the literature. The LC network nevertheless shares attributes with HTP networks, including scale-free connectivity and correlations between interactions, abundance, localization, and expression. We find that essential genes or proteins are enriched for interactions with other essential genes or proteins, suggesting that the global network may be functionally unified. This interconnectivity is supported by a substantial overlap of protein and genetic interactions in the LC dataset. We show that the LC dataset considerably improves the predictive power of network-analysis approaches. The full LC dataset is available at the BioGRID () and SGD () databases. Conclusion Comprehensive datasets of biological interactions derived from the primary literature provide critical benchmarks for HTP methods, augment functional prediction, and reveal system-level attributes of biological networks. PMID:16762047

ANCAC: amino acid, nucleotide, and codon analysis of COGs – a tool for sequence bias analysis in microbial orthologs

PubMed Central

2012-01-01

Background The COG database is the most popular collection of orthologous proteins from many different completely sequenced microbial genomes. Per definition, a cluster of orthologous groups (COG) within this database exclusively contains proteins that most likely achieve the same cellular function. Recently, the COG database was extended by assigning to every protein both the corresponding amino acid and its encoding nucleotide sequence resulting in the NUCOCOG database. This extended version of the COG database is a valuable resource connecting sequence features with the functionality of the respective proteins. Results Here we present ANCAC, a web tool and MySQL database for the analysis of amino acid, nucleotide, and codon frequencies in COGs on the basis of freely definable phylogenetic patterns. We demonstrate the usefulness of ANCAC by analyzing amino acid frequencies, codon usage, and GC-content in a species- or function-specific context. With respect to amino acids we, at least in part, confirm the cognate bias hypothesis by using ANCAC’s NUCOCOG dataset as the largest one available for that purpose thus far. Conclusions Using the NUCOCOG datasets, ANCAC connects taxonomic, amino acid, and nucleotide sequence information with the functional classification via COGs and provides a GUI for flexible mining for sequence-bias. Thereby, to our knowledge, it is the only tool for the analysis of sequence composition in the light of physiological roles and phylogenetic context without requirement of substantial programming-skills. PMID:22958836

CTCF knockout reveals an essential role for this protein during the zebrafish development.

PubMed

Carmona-Aldana, Francisco; Zampedri, Cecilia; Suaste-Olmos, Fernando; Murillo-de-Ozores, Adrián; Guerrero, Georgina; Arzate-Mejía, Rodrigo; Maldonado, Ernesto; Navarro, Rosa; Chimal-Monroy, Jesús; Recillas-Targa, Félix

2018-05-01

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish. Copyright © 2018. Published by Elsevier B.V.

PATtyFams: Protein families for the microbial genomes in the PATRIC database

DOE PAGES

Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; ...

2016-02-08

The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda

PubMed Central

2013-01-01

Background Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. Results In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Conclusions Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles. PMID:24073829

PATtyFams: Protein families for the microbial genomes in the PATRIC database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.

The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

PubMed

Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

2011-05-01

Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Domain architecture conservation in orthologs

PubMed Central

2011-01-01

Background As orthologous proteins are expected to retain function more often than other homologs, they are often used for functional annotation transfer between species. However, ortholog identification methods do not take into account changes in domain architecture, which are likely to modify a protein's function. By domain architecture we refer to the sequential arrangement of domains along a protein sequence. To assess the level of domain architecture conservation among orthologs, we carried out a large-scale study of such events between human and 40 other species spanning the entire evolutionary range. We designed a score to measure domain architecture similarity and used it to analyze differences in domain architecture conservation between orthologs and paralogs relative to the conservation of primary sequence. We also statistically characterized the extents of different types of domain swapping events across pairs of orthologs and paralogs. Results The analysis shows that orthologs exhibit greater domain architecture conservation than paralogous homologs, even when differences in average sequence divergence are compensated for, for homologs that have diverged beyond a certain threshold. We interpret this as an indication of a stronger selective pressure on orthologs than paralogs to retain the domain architecture required for the proteins to perform a specific function. In general, orthologs as well as the closest paralogous homologs have very similar domain architectures, even at large evolutionary separation. The most common domain architecture changes observed in both ortholog and paralog pairs involved insertion/deletion of new domains, while domain shuffling and segment duplication/deletion were very infrequent. Conclusions On the whole, our results support the hypothesis that function conservation between orthologs demands higher domain architecture conservation than other types of homologs, relative to primary sequence conservation. This supports the notion that orthologs are functionally more similar than other types of homologs at the same evolutionary distance. PMID:21819573

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

When the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases

PubMed Central

2011-01-01

Background Comprehensive understanding of molecular mechanisms underlying viral infection is a major challenge towards the discovery of new antiviral drugs and susceptibility factors of human diseases. New advances in the field are expected from systems-level modelling and integration of the incessant torrent of high-throughput "-omics" data. Results Here, we describe the Human Infectome protein interaction Network, a novel systems virology model of a virtual virus-infected human cell concerning 110 viruses. This in silico model was applied to comprehensively explore the molecular relationships between viruses and their associated diseases. This was done by merging virus-host and host-host physical protein-protein interactomes with the set of genes essential for viral replication and involved in human genetic diseases. This systems-level approach provides strong evidence that viral proteomes target a wide range of functional and inter-connected modules of proteins as well as highly central and bridging proteins within the human interactome. The high centrality of targeted proteins was correlated to their essentiality for viruses' lifecycle, using functional genomic RNAi data. A stealth-attack of viruses on proteins bridging cellular functions was demonstrated by simulation of cellular network perturbations, a property that could be essential in the molecular aetiology of some human diseases. Networking the Human Infectome and Diseasome unravels the connectivity of viruses to a wide range of diseases and profiled molecular basis of Hepatitis C Virus-induced diseases as well as 38 new candidate genetic predisposition factors involved in type 1 diabetes mellitus. Conclusions The Human Infectome and Diseasome Networks described here provide a unique gateway towards the comprehensive modelling and analysis of the systems level properties associated to viral infection as well as candidate genes potentially involved in the molecular aetiology of human diseases. PMID:21255393

CABS-flex predictions of protein flexibility compared with NMR ensembles

PubMed Central

Jamroz, Michal; Kolinski, Andrzej; Kmiecik, Sebastian

2014-01-01

Motivation: Identification of flexible regions of protein structures is important for understanding of their biological functions. Recently, we have developed a fast approach for predicting protein structure fluctuations from a single protein model: the CABS-flex. CABS-flex was shown to be an efficient alternative to conventional all-atom molecular dynamics (MD). In this work, we evaluate CABS-flex and MD predictions by comparison with protein structural variations within NMR ensembles. Results: Based on a benchmark set of 140 proteins, we show that the relative fluctuations of protein residues obtained from CABS-flex are well correlated to those of NMR ensembles. On average, this correlation is stronger than that between MD and NMR ensembles. In conclusion, CABS-flex is useful and complementary to MD in predicting protein regions that undergo conformational changes as well as the extent of such changes. Availability and implementation: The CABS-flex is freely available to all users at http://biocomp.chem.uw.edu.pl/CABSflex. Contact: sekmi@chem.uw.edu.pl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24735558

A proteomic insight into vitellogenesis during tick ovary maturation.

PubMed

Xavier, Marina Amaral; Tirloni, Lucas; Pinto, Antônio F M; Diedrich, Jolene K; Yates, John R; Mulenga, Albert; Logullo, Carlos; da Silva Vaz, Itabajara; Seixas, Adriana; Termignoni, Carlos

2018-03-16

Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.

CABS-flex predictions of protein flexibility compared with NMR ensembles.

PubMed

Jamroz, Michal; Kolinski, Andrzej; Kmiecik, Sebastian

2014-08-01

Identification of flexible regions of protein structures is important for understanding of their biological functions. Recently, we have developed a fast approach for predicting protein structure fluctuations from a single protein model: the CABS-flex. CABS-flex was shown to be an efficient alternative to conventional all-atom molecular dynamics (MD). In this work, we evaluate CABS-flex and MD predictions by comparison with protein structural variations within NMR ensembles. Based on a benchmark set of 140 proteins, we show that the relative fluctuations of protein residues obtained from CABS-flex are well correlated to those of NMR ensembles. On average, this correlation is stronger than that between MD and NMR ensembles. In conclusion, CABS-flex is useful and complementary to MD in predicting protein regions that undergo conformational changes as well as the extent of such changes. The CABS-flex is freely available to all users at http://biocomp.chem.uw.edu.pl/CABSflex. sekmi@chem.uw.edu.pl Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

Immunopurification of adenomatous polyposis coli (APC) proteins

PubMed Central

2013-01-01

Background The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. Results In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1–2843) and cancer-associated, truncated APC proteins, APC(1–1638) and APC(1–1311) were produced in Sf9 insect cells. Conclusions Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein. PMID:24156781

Natural variation in floral nectar proteins of two Nicotiana attenuata accessions

PubMed Central

2013-01-01

Background Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. Results We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. Conclusions Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions. PMID:23848992

New Stable Isotope Method to Measure Protein Digestibility and Response to Pancreatic Enzyme Intake in Cystic Fibrosis

PubMed Central

Engelen, M.P.K.J.; Com, G.; Anderson, P.J.; Deutz, N.E.P.

2015-01-01

Background & Aims Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. Objective To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. Design In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 hours by adding 15N-labeled spirulina protein and L-[ring-2H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [15N]PHE to [2H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-13C-5,5-2H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Results Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P<0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n=10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Conclusion Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. PMID:24268783

Integration of somatic mutation, expression and functional data reveals potential driver genes predictive of breast cancer survival.

PubMed

Suo, Chen; Hrydziuszko, Olga; Lee, Donghwan; Pramana, Setia; Saputra, Dhany; Joshi, Himanshu; Calza, Stefano; Pawitan, Yudi

2015-08-15

Genome and transcriptome analyses can be used to explore cancers comprehensively, and it is increasingly common to have multiple omics data measured from each individual. Furthermore, there are rich functional data such as predicted impact of mutations on protein coding and gene/protein networks. However, integration of the complex information across the different omics and functional data is still challenging. Clinical validation, particularly based on patient outcomes such as survival, is important for assessing the relevance of the integrated information and for comparing different procedures. An analysis pipeline is built for integrating genomic and transcriptomic alterations from whole-exome and RNA sequence data and functional data from protein function prediction and gene interaction networks. The method accumulates evidence for the functional implications of mutated potential driver genes found within and across patients. A driver-gene score (DGscore) is developed to capture the cumulative effect of such genes. To contribute to the score, a gene has to be frequently mutated, with high or moderate mutational impact at protein level, exhibiting an extreme expression and functionally linked to many differentially expressed neighbors in the functional gene network. The pipeline is applied to 60 matched tumor and normal samples of the same patient from The Cancer Genome Atlas breast-cancer project. In clinical validation, patients with high DGscores have worse survival than those with low scores (P = 0.001). Furthermore, the DGscore outperforms the established expression-based signatures MammaPrint and PAM50 in predicting patient survival. In conclusion, integration of mutation, expression and functional data allows identification of clinically relevant potential driver genes in cancer. The documented pipeline including annotated sample scripts can be found in http://fafner.meb.ki.se/biostatwiki/driver-genes/. yudi.pawitan@ki.se Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Functional specialization in nucleotide sugar transporters occurred through differentiation of the gene cluster EamA (DUF6) before the radiation of Viridiplantae

PubMed Central

2011-01-01

Background The drug/metabolite transporter superfamily comprises a diversity of protein domain families with multiple functions including transport of nucleotide sugars. Drug/metabolite transporter domains are contained in both solute carrier families 30, 35 and 39 proteins as well as in acyl-malonyl condensing enzyme proteins. In this paper, we present an evolutionary analysis of nucleotide sugar transporters in relation to the entire superfamily of drug/metabolite transporters that considers crucial intra-protein duplication events that have shaped the transporters. We use a method that combines the strengths of hidden Markov models and maximum likelihood to find relationships between drug/metabolite transporter families, and branches within families. Results We present evidence that the triose-phosphate transporters, domain unknown function 914, uracil-diphosphate glucose-N-acetylglucosamine, and nucleotide sugar transporter families have evolved from a domain duplication event before the radiation of Viridiplantae in the EamA family (previously called domain unknown function 6). We identify previously unknown branches in the solute carrier 30, 35 and 39 protein families that emerged simultaneously as key physiological developments after the radiation of Viridiplantae, including the "35C/E" branch of EamA, which formed in the lineage of T. adhaerens (Animalia). We identify a second cluster of DMTs, called the domain unknown function 1632 cluster, which has non-cytosolic N- and C-termini, and thus appears to have been formed from a different domain duplication event. We identify a previously uncharacterized motif, G-X(6)-G, which is overrepresented in the fifth transmembrane helix of C-terminal domains. We present evidence that the family called fatty acid elongases are homologous to transporters, not enzymes as had previously been thought. Conclusions The nucleotide sugar transporters families were formed through differentiation of the gene cluster EamA (domain unknown function 6) before Viridiplantae, showing for the first time the significance of EamA. PMID:21569384

Protein structure based prediction of catalytic residues

PubMed Central

2013-01-01

Background Worldwide structural genomics projects continue to release new protein structures at an unprecedented pace, so far nearly 6000, but only about 60% of these proteins have any sort of functional annotation. Results We explored a range of features that can be used for the prediction of functional residues given a known three-dimensional structure. These features include various centrality measures of nodes in graphs of interacting residues: closeness, betweenness and page-rank centrality. We also analyzed the distance of functional amino acids to the general center of mass (GCM) of the structure, relative solvent accessibility (RSA), and the use of relative entropy as a measure of sequence conservation. From the selected features, neural networks were trained to identify catalytic residues. We found that using distance to the GCM together with amino acid type provide a good discriminant function, when combined independently with sequence conservation. Using an independent test set of 29 annotated protein structures, the method returned 411 of the initial 9262 residues as the most likely to be involved in function. The output 411 residues contain 70 of the annotated 111 catalytic residues. This represents an approximately 14-fold enrichment of catalytic residues on the entire input set (corresponding to a sensitivity of 63% and a precision of 17%), a performance competitive with that of other state-of-the-art methods. Conclusions We found that several of the graph based measures utilize the same underlying feature of protein structures, which can be simply and more effectively captured with the distance to GCM definition. This also has the added the advantage of simplicity and easy implementation. Meanwhile sequence conservation remains by far the most influential feature in identifying functional residues. We also found that due the rapid changes in size and composition of sequence databases, conservation calculations must be recalibrated for specific reference databases. PMID:23433045

Buffer Effects in the Solubility, Nucleation and Growth of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Gibson, Ursula J.

1999-01-01

The growth of protein crystals is important for determination of their three-dimensional structure, which relates to their biochemical functions and to the practical goal of designing pharmaceuticals to modify that function. While many proteins have been successfully crystallized by a variety of methods, there is still limited understanding of the process of nucleation and growth of even the simplest proteins. Chicken egg-white lysozyme (CEWL) is readily crystallized under a variety of conditions, and studies underway at MSFC are designed to elucidate the mechanisms by which the crystals nucleate and grow. We have investigated the effect of buffer choice on the solubility, nucleation and growth of CEWL. CEWL was purified by dialysis against a .05M phosphate buffer and chromatographic separation from contaminants in a sepharose column. Solubility studies were made as a function of buffer concentration for phosphate and formate buffers, and the nucleation and growth of crystals at 10 C was studied as a function of pH for oxalate, succinate, formate, butyrate, carbonate, phosphate and acetate buffer solutions. The solubility data support the conclusion that there is a solubility minimum as a function of buffer concentration for amphiphilic molecules, while no minimum is observed for a phosphate buffer. Nucleation is suppressed at pH greater than pKa for all buffers except phosphate. The aspect ratio of the (110) faces is shown to be a function of crystal size, rather than pH.

When the Lowest Energy Does Not Induce Native Structures: Parallel Minimization of Multi-Energy Values by Hybridizing Searching Intelligences

PubMed Central

Lü, Qiang; Xia, Xiao-Yan; Chen, Rong; Miao, Da-Jun; Chen, Sha-Sha; Quan, Li-Jun; Li, Hai-Ou

2012-01-01

Background Protein structure prediction (PSP), which is usually modeled as a computational optimization problem, remains one of the biggest challenges in computational biology. PSP encounters two difficult obstacles: the inaccurate energy function problem and the searching problem. Even if the lowest energy has been luckily found by the searching procedure, the correct protein structures are not guaranteed to obtain. Results A general parallel metaheuristic approach is presented to tackle the above two problems. Multi-energy functions are employed to simultaneously guide the parallel searching threads. Searching trajectories are in fact controlled by the parameters of heuristic algorithms. The parallel approach allows the parameters to be perturbed during the searching threads are running in parallel, while each thread is searching the lowest energy value determined by an individual energy function. By hybridizing the intelligences of parallel ant colonies and Monte Carlo Metropolis search, this paper demonstrates an implementation of our parallel approach for PSP. 16 classical instances were tested to show that the parallel approach is competitive for solving PSP problem. Conclusions This parallel approach combines various sources of both searching intelligences and energy functions, and thus predicts protein conformations with good quality jointly determined by all the parallel searching threads and energy functions. It provides a framework to combine different searching intelligence embedded in heuristic algorithms. It also constructs a container to hybridize different not-so-accurate objective functions which are usually derived from the domain expertise. PMID:23028708

Bioinformatics analysis of disordered proteins in prokaryotes

PubMed Central

2011-01-01

Background A significant number of proteins have been shown to be intrinsically disordered, meaning that they lack a fixed 3 D structure or contain regions that do not posses a well defined 3 D structure. It has also been proven that a protein's disorder content is related to its function. We have performed an exhaustive analysis and comparison of the disorder content of proteins from prokaryotic organisms (i.e., superkingdoms Archaea and Bacteria) with respect to functional categories they belong to, i.e., Clusters of Orthologous Groups of proteins (COGs) and groups of COGs-Cellular processes (Cp), Information storage and processing (Isp), Metabolism (Me) and Poorly characterized (Pc). We also analyzed the disorder content of proteins with respect to various genomic, metabolic and ecological characteristics of the organism they belong to. We used correlations and association rule mining in order to identify the most confident associations between specific modalities of the characteristics considered and disorder content. Results Bacteria are shown to have a somewhat higher level of protein disorder than archaea, except for proteins in the Me functional group. It is demonstrated that the Isp and Cp functional groups in particular (L-repair function and N-cell motility and secretion COGs of proteins in specific) possess the highest disorder content, while Me proteins, in general, posses the lowest. Disorder fractions have been confirmed to have the lowest level for the so-called order-promoting amino acids and the highest level for the so-called disorder promoters. For each pair of organism characteristics, specific modalities are identified with the maximum disorder proteins in the corresponding organisms, e.g., high genome size-high GC content organisms, facultative anaerobic-low GC content organisms, aerobic-high genome size organisms, etc. Maximum disorder in archaea is observed for high GC content-low genome size organisms, high GC content-facultative anaerobic or aquatic or mesophilic organisms, etc. Maximum disorder in bacteria is observed for high GC content-high genome size organisms, high genome size-aerobic organisms, etc. Some of the most reliable association rules mined establish relationships between high GC content and high protein disorder, medium GC content and both medium and low protein disorder, anaerobic organisms and medium protein disorder, Gammaproteobacteria and low protein disorder, etc. A web site Prokaryote Disorder Database has been designed and implemented at the address http://bioinfo.matf.bg.ac.rs/disorder, which contains complete results of the analysis of protein disorder performed for 296 prokaryotic completely sequenced genomes. Conclusions Exhaustive disorder analysis has been performed by functional classes of proteins, for a larger dataset of prokaryotic organisms than previously done. Results obtained are well correlated to those previously published, with some extension in the range of disorder level and clear distinction between functional classes of proteins. Wide correlation and association analysis between protein disorder and genomic and ecological characteristics has been performed for the first time. The results obtained give insight into multi-relationships among the characteristics and protein disorder. Such analysis provides for better understanding of the evolutionary process and may be useful for taxon determination. The main drawback of the approach is the fact that the disorder considered has been predicted and not experimentally established. PMID:21366926

Overexpression of FOXA1 inhibits cell proliferation and EMT of human gastric cancer AGS cells.

PubMed

Lin, Mengxin; Pan, Jie; Chen, Qiang; Xu, Zongbin; Lin, Xiaoyan; Shi, Chunmei

2018-02-05

The lack of effective medical treatment for advanced stages of gastric cancer mainly contributes to the high mortality rate. The association of forkhead box protein A1 (FOXA1) with tumor progression has been reported in different human cancers. However, the function of FOXA1 in gastric cancer is largely unknown. In the present study, FOXA1 protein showed a significant reduction in gastric cancer samples comparing with matched control samples. In addition, the higher expression of FOXA1 in transcription level was observed in gastric cancer cell lines as compared with that in normal gastric cell line, while the contrary result was observed in protein level. Then we studied the effects of FOXA1 on gastric cancer cells in vitro and in vivo based on FOXA1-overexpression AGS cells. We found that up-regulation of FOXA1 was notably inhibited the cell proliferation and tumor formation, and induced cell apoptosis. Moreover, overexpression of FOXA1 was able to increase the E-cadherin protein level and decreased the Vimentin protein level, which implicates that FOXA1 probably plays as an inhibitor of epithelial mesenchymal transition. In conclusion, these data suggests that FOXA1 may function as a novel anti-oncogene in gastric cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

PubMed Central

Yama, Tomonari; Ochi, Arisa; Suto, Takuro; Hirasaka, Katsuya; Teshima-Kondo, Shigetada; Okumura, Yuushi; Oarada, Motoko; Choi, Inho; Mukai, Rie; Terao, Junji

2013-01-01

Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice. PMID:23762056

Structural biology of intrinsically disordered proteins: Revisiting unsolved mysteries.

PubMed

Sigalov, Alexander B

2016-06-01

The emergence of intrinsically disordered proteins (IDPs) has challenged the classical protein structure-function paradigm by introducing a new paradigm of "coupled binding and folding". This paradigm suggests that IDPs fold upon binding to their partners. Further studies, however, revealed a novel and previously unrecognized phenomenon of "uncoupled binding and folding" suggesting that IDPs do not necessarily fold upon interaction with their lipid and protein partners. The complex and often unusual biophysics of IDPs makes structural characterization of these proteins and their complexes not only challenging but often resulting in opposite conclusions. For this reason, some crucial questions in this field remain unsolved for well over a decade. Considering an important role of IDPs in cellular regulation, signaling and control in health and disease, more efforts are needed to solve these mysteries. Here, I focus on two long-standing contradictions in the literature concerning dimerization and membrane-binding activities of IDPs. Molecular explanation of these discrepancies is provided. I also demonstrate how resolution of these critical issues in the field of IDPs results in our expanded understanding of cell function and has multiple applications in biology and medicine. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

PubMed

Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

PubMed Central

2005-01-01

Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. PMID:16313672

A discriminative method for family-based protein remote homology detection that combines inductive logic programming and propositional models

PubMed Central

2011-01-01

Background Remote homology detection is a hard computational problem. Most approaches have trained computational models by using either full protein sequences or multiple sequence alignments (MSA), including all positions. However, when we deal with proteins in the "twilight zone" we can observe that only some segments of sequences (motifs) are conserved. We introduce a novel logical representation that allows us to represent physico-chemical properties of sequences, conserved amino acid positions and conserved physico-chemical positions in the MSA. From this, Inductive Logic Programming (ILP) finds the most frequent patterns (motifs) and uses them to train propositional models, such as decision trees and support vector machines (SVM). Results We use the SCOP database to perform our experiments by evaluating protein recognition within the same superfamily. Our results show that our methodology when using SVM performs significantly better than some of the state of the art methods, and comparable to other. However, our method provides a comprehensible set of logical rules that can help to understand what determines a protein function. Conclusions The strategy of selecting only the most frequent patterns is effective for the remote homology detection. This is possible through a suitable first-order logical representation of homologous properties, and through a set of frequent patterns, found by an ILP system, that summarizes essential features of protein functions. PMID:21429187

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging.

PubMed

Park, Sung-Jun; Gavrilova, Oksana; Brown, Alexandra L; Soto, Jamie E; Bremner, Shannon; Kim, Jeonghan; Xu, Xihui; Yang, Shutong; Um, Jee-Hyun; Koch, Lauren G; Britton, Steven L; Lieber, Richard L; Philp, Andrew; Baar, Keith; Kohama, Steven G; Abel, E Dale; Kim, Myung K; Chung, Jay H

2017-05-02

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity. Published by Elsevier Inc.

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

PubMed Central

Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

2012-01-01

Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

Not so pseudo: the evolutionary history of protein phosphatase 1 regulatory subunit 2 and related pseudogenes

PubMed Central

2013-01-01

Background Pseudogenes are traditionally considered “dead” genes, therefore lacking biological functions. This view has however been challenged during the last decade. This is the case of the Protein phosphatase 1 regulatory subunit 2 (PPP1R2) or inhibitor-2 gene family, for which several incomplete copies exist scattered throughout the genome. Results In this study, the pseudogenization process of PPP1R2 was analyzed. Ten PPP1R2-related pseudogenes (PPP1R2P1-P10), highly similar to PPP1R2, were retrieved from the human genome assembly present in the databases. The phylogenetic analysis of mammalian PPP1R2 and related pseudogenes suggested that PPP1R2P7 and PPP1R2P9 retroposons appeared before the great mammalian radiation, while the remaining pseudogenes are primate-specific and retroposed at different times during Primate evolution. Although considered inactive, four of these pseudogenes seem to be transcribed and possibly possess biological functions. Given the role of PPP1R2 in sperm motility, the presence of these proteins was assessed in human sperm, and two PPP1R2-related proteins were detected, PPP1R2P3 and PPP1R2P9. Signatures of negative and positive selection were also detected in PPP1R2P9, further suggesting a role as a functional protein. Conclusions The results show that contrary to initial observations PPP1R2-related pseudogenes are not simple bystanders of the evolutionary process but may rather be at the origin of genes with novel functions. PMID:24195737

Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation

PubMed Central

Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Hernandez, Joseph; Potluri, Vani; Wang, Liguo; Li, Wei; Lemay, Danielle G.

2013-01-01

Background Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. Methodology/Principal Findings Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. Conclusions/Significance A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland. PMID:23301053

Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

PubMed Central

Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

2015-01-01

Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

PubMed Central

2012-01-01

Background Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G) or reduced (TaxS240P) transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting, respectively, after transfecting Tax proteins into bovine cells and human HeLa cells. Conclusion A comparative analysis of wild-type and mutant Tax proteins indicates that Tax protein exerts a significant impact on cellular functions as diverse as transcription, signal transduction, cell growth, stress response and immune response. Importantly, our study is the first report that shows the extent to which BLV Tax regulates the innate immune response. PMID:22455445

Machine-learning scoring functions for identifying native poses of ligands docked to known and novel proteins

PubMed Central

2015-01-01

Background Molecular docking is a widely-employed method in structure-based drug design. An essential component of molecular docking programs is a scoring function (SF) that can be used to identify the most stable binding pose of a ligand, when bound to a receptor protein, from among a large set of candidate poses. Despite intense efforts in developing conventional SFs, which are either force-field based, knowledge-based, or empirical, their limited docking power (or ability to successfully identify the correct pose) has been a major impediment to cost-effective drug discovery. Therefore, in this work, we explore a range of novel SFs employing different machine-learning (ML) approaches in conjunction with physicochemical and geometrical features characterizing protein-ligand complexes to predict the native or near-native pose of a ligand docked to a receptor protein's binding site. We assess the docking accuracies of these new ML SFs as well as those of conventional SFs in the context of the 2007 PDBbind benchmark dataset on both diverse and homogeneous (protein-family-specific) test sets. Further, we perform a systematic analysis of the performance of the proposed SFs in identifying native poses of ligands that are docked to novel protein targets. Results and conclusion We find that the best performing ML SF has a success rate of 80% in identifying poses that are within 1 Å root-mean-square deviation from the native poses of 65 different protein families. This is in comparison to a success rate of only 70% achieved by the best conventional SF, ASP, employed in the commercial docking software GOLD. In addition, the proposed ML SFs perform better on novel proteins that they were never trained on before. We also observed steady gains in the performance of these scoring functions as the training set size and number of features were increased by considering more protein-ligand complexes and/or more computationally-generated poses for each complex. PMID:25916860

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens

PubMed Central

Gaudin, Jean-Charles; Patil, Sarita; Steinbrecher, Johanna; Matsunaga, Kayoko; Teshima, Reiko; Sakai, Shinobu; Larré, Colette; Denery-Papini, Sandra

2017-01-01

Background Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found. Objectives In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients’ IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs. Methods Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients’ IgE. Results Acid-HWPs with medium (30%) and high (50–60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients’ IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay. Conclusion Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins. PMID:29117222

Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

PubMed Central

Mouro-Chanteloup, Isabelle; Cochet, Sylvie; Chami, Mohamed; Genetet, Sandrine; Zidi-Yahiaoui, Nedjma; Engel, Andreas; Colin, Yves; Bertrand, Olivier; Ripoche, Pierre

2010-01-01

Background Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. Methodology/Principal Findings An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C12E8 detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. Conclusions/Significance This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red blood cells endogenously expressing RhAG (2.18×10−3 µm3.s−1). The major finding of this study is that RhCG protein is active as an NH3 channel and that this function does not require any protein partner. PMID:20126667

Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

PubMed Central

2011-01-01

Background The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H+-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na+-dependent Pi (NaPi) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. Results We show that the human PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic for all PiT family members. A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR254-V483), was found to be a fully functional Pi transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL183-V483) did also support Pi transport albeit at very low levels. Conclusions The results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship. PMID:21586110

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

The RLK/Pelle family of kinases.

PubMed

Gish, Lindsey A; Clark, Steven E

2011-04-01

The RLK/Pelle class of proteins kinases is composed of over 600 members in Arabidopsis. Many of the proteins in this family are receptor-like kinases (RLK), while others have lost their extracellular domains and are found as cytoplasmic kinases. Proteins in this family that are RLKs have a variety of extracellular domains that drive function in a large number of processes, from cell wall interactions to disease resistance to developmental control. This review will briefly cover the major subclasses of RLK/Pelle proteins and their roles. In addition, two specific groups on RLKs will be discussed in detail, relating recent findings in Arabidopsis and how well these conclusions have been able to be translated to agronomically important species. Finally, some details on kinase activity and signal transduction will be addressed, along with the mystery of RLK/Pelle members lacking kinase enzymatic activity. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

PubMed Central

Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

2010-01-01

Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family.

PubMed

Johnson, Glynis; Moore, Samuel W

2013-09-01

Short linear motifs confer evolutionary flexibility on proteins as they can be added with relative ease allowing the acquisition of new functions. Such motifs may mediate a variety of signalling functions. The adhesion-mediating Leu-Arg-Glu (LRE) motif is enriched in laminin beta 2, and has been observed in other proteins, including members of the carboxylesterase/cholinesterase family. It acts as a stop signal for growing axons in the developing neuromuscular junction, binding to the voltage-gated calcium channel. In this bioinformatic analysis, we have investigated the presence of the motif in proteins of the neuromuscular junction, and have also examined its structural position and potential for ligand interaction, as well as phylogenetic conservation, in the carboxylesterase/cholinesterase family. The motif was observed to occur with a significantly higher frequency than expected in the UniProt/Swiss-Prot database, as well as in four individual species (human, mouse, Caenorhabditis elegans and Drosophila melanogaster). Examination of its presence in neuromuscular junction proteins showed it to be enriched in certain proteins of the synaptic basement membrane, including laminin, agrin, acetylcholinesterase and tenascin. A highly significant enrichment was observed in cytoskeletal proteins, particularly intermediate filament proteins and members of the spectrin family. In the carboxylesterase/cholinesterase family, the motif was observed in four conserved positions in the protein structure. It is present in the majority of mammalian acetylcholinesterases, as well as acetylcholinesterases from electric fish and a number of invertebrates. In insects, it is present in the ace-2, rather than in the synaptic ace-1, enzyme. It is also observed in the cholinesterase-like adhesion molecules (neuroligins, neurotactin and glutactin). It is never seen in butyrylcholinesterases, which do not mediate cell adhesion. In conclusion, the significant enrichment of the motif in certain classes of protein, as well as its conserved presence and structural positioning in one protein family, suggests that it has specific functions both in cell adhesion in the neuromuscular junction and in maintaining the structural integrity of the cytoskeleton. Copyright © 2013 Elsevier Inc. All rights reserved.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

PubMed

Brito, Mayara F; Auler, Patrícia A; Tavares, Guilherme C; Rezende, Cristiana P; Almeida, Gabriel M F; Pereira, Felipe L; Leal, Carlos A G; Moura, Arlindo de Alencar; Figueiredo, Henrique C P; Henry, Marc

2018-06-11

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728. © 2018 Blackwell Verlag GmbH.

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

PubMed Central

2009-01-01

Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion. PMID:20034395

Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells.

PubMed

Yang, Shu; Jin, Hong; Zhao, Zhigang

2018-04-23

Objective The blood-brain barrier (BBB), regulating brain homeostasis and limiting the entry of most drugs, is characterized by intercellular tight junctions and the presence of transporters. In this study, the paracellular tightness and functional expression of efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were evaluated in mouse brain immortalized cell line bEnd3 to prove it as a useful BBB-mimicking system for biological and pharmacological research. Methods The presence of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 were validated by RT-PCR and Western blot. The tightness of bEnd3 monolayers was evaluated by measuring the permeability of hydrophilic marker Lucifer yellow. The P-gp functionality was identified by intracellular uptake assay using Rhodamine 123 (R123) as P-gp substrate and verapamil as P-gp inhibitor. The BCRP functionality was identified by flow cytometric analysis of mitoxantrone accumulation and fluorescence microscopic analysis of Hoechst 33342 accumulation using Ko-143 as BCRP inhibitor. Results The bEnd3 cells demonstrated the expression of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 at mRNA and protein levels. The permeability coefficient of Lucifer yellow was 1.3 ± 0.13 × 10 -3  cm/min, indicating the moderate paracellular tightness barrier formed by bEnd3 cells. The verapamil induced a higher cellular uptake of Rhodamine 123, and Ko-143 significantly elevated cellular accumulation of mitoxantrone and Hoechst 33342, suggesting the P-gp and BCRP functionality shown by bEnd3 cells. Conclusions The bEnd3 cell line represents a useful in vitro tool for studying BBB characteristics and drug transport mechanisms at the BBB.

The adaptive evolution of the mammalian mitochondrial genome

PubMed Central

da Fonseca, Rute R; Johnson, Warren E; O'Brien, Stephen J; Ramos, Maria João; Antunes, Agostinho

2008-01-01

Background The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures. Results Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome b in species with more-specialized metabolic requirements (such as adaptation to low energy diet or large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas). Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome c oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F0, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas. Conclusion Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation. PMID:18318906

Platelet ERK5 is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion

PubMed Central

Cameron, Scott J.; Ture, Sara K.; Mickelsen, Deanne; Chakrabarti, Enakshi; Modjeski, Kristina L.; McNitt, Scott; Seaberry, Micheal; Field, David J.; Le, Nhat-Tu; Abe, Jun-ichi; Morrell, Craig N.

2015-01-01

Background Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes (ACS). Compared to platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species (ROS) rich environments may not be the same as in normal healthy conditions. Extracellular Regulated Protein Kinase 5 (ERK5) is a Mitogen Activated Protein Kinase (MAPK) family member activated in hypoxic, ROS rich environments, and in response to receptor signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells following ischemia. We present evidence that platelets express ERK5 and platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent ROS mediated mechanisms in ischemic myocardium. Methods and Results Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and platelet specific ERK5−/− mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5 regulated proteins is reduced in ERK5−/− platelets post-MI. Conclusions ERK5 functions as a platelet activator in ischemic conditions and platelet ERK5 maintains the expression of some platelet proteins following MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment. PMID:25934838

Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations.

PubMed

Elvenes, Julianne; Sjøttem, Eva; Holm, Turid; Bjørkøy, Geir; Johansen, Terje

2010-12-01

The transcription factor Pax6 is crucial for the embryogenesis of multiple organs, including the eyes, parts of the brain and the pancreas. Mutations in one allele of PAX6 lead to eye diseases including Peter's anomaly and aniridia. Here, we use fluorescence recovery after photobleaching to show that Pax6 and also other Pax family proteins display a strikingly low nuclear mobility compared to other transcriptional regulators. For Pax6, the slow mobility is largely due to the presence of two DNA-binding domains, but protein-protein interactions also contribute. Consistently, the subnuclear localization of Pax6 suggests that it interacts preferentially with chromatin-rich territories. Some aniridia-causing missense mutations in Pax6 have impaired DNA-binding affinity. Interestingly, when these mutants were analyzed by FRAP, they displayed a pronounced increased mobility compared to wild-type Pax6. Hence, our results support the conclusion that disease mutations result in proteins with impaired function because of altered DNA- and protein-interaction capabilities.

C-terminus of HSC70-Interacting Protein (CHIP) Inhibits Adipocyte Differentiation via Ubiquitin- and Proteasome-Mediated Degradation of PPARγ

PubMed Central

Kim, Jung-Hoon; Shin, Soyeon; Seo, Jinho; Lee, Eun-Woo; Jeong, Manhyung; Lee, Min-sik; Han, Hyun-Ji; Song, Jaewhan

2017-01-01

PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis. PMID:28059128

Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3

PubMed Central

2014-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. Results The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. Conclusions ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function. PMID:24998259

The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury

PubMed Central

Cramer, Samuel W; Baggott, Christopher; Cain, John; Tilghman, Jessica; Allcock, Bradley; Miranpuri, Gurwattan; Rajpal, Sharad; Sun, Dandan; Resnick, Daniel

2008-01-01

Background Altered Cl- homeostasis and GABAergic function are associated with nociceptive input hypersensitivity. This study investigated the role of two major intracellular Cl- regulatory proteins, Na+-K+-Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 2 (KCC2), in neuropathic pain following spinal cord injury (SCI). Results Sprague-Dawley rats underwent a contusive SCI at T9 using the MASCIS impactor. The rats developed hyperalgesia between days 21 and 42 post-SCI. Thermal hyperalgesia (TH) was determined by a decrease in hindpaw thermal withdrawal latency time (WLT) between days 21 and 42 post-SCI. Rats with TH were then treated with either vehicle (saline containing 0.25% NaOH) or NKCC1 inhibitor bumetanide (BU, 30 mg/kg, i.p.) in vehicle. TH was then re-measured at 1 h post-injection. Administration of BU significantly increased the mean WLT in rats (p < 0.05). The group administered with the vehicle alone showed no anti-hyperalgesic effects. Moreover, an increase in NKCC1 protein expression occurred in the lesion epicenter of the spinal cord during day 2–14 post-SCI and peaked on day 14 post-SCI (p < 0.05). Concurrently, a down-regulation of KCC2 protein was detected during day 2–14 post-SCI. The rats with TH exhibited a sustained loss of KCC2 protein during post-SCI days 21–42. No significant changes of these proteins were detected in the rostral region of the spinal cord. Conclusion Taken together, expression of NKCC1 and KCC2 proteins was differentially altered following SCI. The anti-hyperalgesic effect of NKCC1 inhibition suggests that normal or elevated NKCC1 function and loss of KCC2 function play a role in the development and maintenance of SCI-induced neuropathic pain. PMID:18799000

Delayed Administration of Bone Marrow Mesenchymal Stem Cell Conditioned Medium Significantly Improves Outcome After Retinal Ischemia in Rats

PubMed Central

Dreixler, John C.; Poston, Jacqueline N.; Balyasnikova, Irina; Shaikh, Afzhal R.; Tupper, Kelsey Y.; Conway, Sineadh; Boddapati, Venkat; Marcet, Marcus M.; Lesniak, Maciej S.; Roth, Steven

2014-01-01

Purpose. Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. Methods. Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-μm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. Results. Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. Conclusions. By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect. PMID:24699381

Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

PubMed Central

2014-01-01

Background The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays. Results We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed. Conclusions It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events. PMID:24594072

Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome

PubMed Central

2013-01-01

Background The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated “omics” approaches. Results The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization. Conclusions The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity. PMID:23394930

Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops—A Proteomic Perspective

PubMed Central

Kosová, Klára; Vítámvás, Pavel; Urban, Milan Oldřich; Klíma, Miroslav; Roy, Amitava; Prášil, Ilja Tom

2015-01-01

Abiotic stress factors, especially low temperatures, drought, and salinity, represent the major constraints limiting agricultural production in temperate climate. Under the conditions of global climate change, the risk of damaging effects of abiotic stresses on crop production increases. Plant stress response represents an active process aimed at an establishment of novel homeostasis under altered environmental conditions. Proteins play a crucial role in plant stress response since they are directly involved in shaping the final phenotype. In the review, results of proteomic studies focused on stress response of major crops grown in temperate climate including cereals: common wheat (Triticum aestivum), durum wheat (Triticum durum), barley (Hordeum vulgare), maize (Zea mays); leguminous plants: alfalfa (Medicago sativa), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum); oilseed rape (Brassica napus); potato (Solanum tuberosum); tobacco (Nicotiana tabaccum); tomato (Lycopersicon esculentum); and others, to a wide range of abiotic stresses (cold, drought, salinity, heat, imbalances in mineral nutrition and heavy metals) are summarized. The dynamics of changes in various protein functional groups including signaling and regulatory proteins, transcription factors, proteins involved in protein metabolism, amino acid metabolism, metabolism of several stress-related compounds, proteins with chaperone and protective functions as well as structural proteins (cell wall components, cytoskeleton) are briefly overviewed. Attention is paid to the differences found between differentially tolerant genotypes. In addition, proteomic studies aimed at proteomic investigation of multiple stress factors are discussed. In conclusion, contribution of proteomic studies to understanding the complexity of crop response to abiotic stresses as well as possibilities to identify and utilize protein markers in crop breeding processes are discussed. PMID:26340626

FPGA accelerator for protein secondary structure prediction based on the GOR algorithm

PubMed Central

2011-01-01

Background Protein is an important molecule that performs a wide range of functions in biological systems. Recently, the protein folding attracts much more attention since the function of protein can be generally derived from its molecular structure. The GOR algorithm is one of the most successful computational methods and has been widely used as an efficient analysis tool to predict secondary structure from protein sequence. However, the execution time is still intolerable with the steep growth in protein database. Recently, FPGA chips have emerged as one promising application accelerator to accelerate bioinformatics algorithms by exploiting fine-grained custom design. Results In this paper, we propose a complete fine-grained parallel hardware implementation on FPGA to accelerate the GOR-IV package for 2D protein structure prediction. To improve computing efficiency, we partition the parameter table into small segments and access them in parallel. We aggressively exploit data reuse schemes to minimize the need for loading data from external memory. The whole computation structure is carefully pipelined to overlap the sequence loading, computing and back-writing operations as much as possible. We implemented a complete GOR desktop system based on an FPGA chip XC5VLX330. Conclusions The experimental results show a speedup factor of more than 430x over the original GOR-IV version and 110x speedup over the optimized version with multi-thread SIMD implementation running on a PC platform with AMD Phenom 9650 Quad CPU for 2D protein structure prediction. However, the power consumption is only about 30% of that of current general-propose CPUs. PMID:21342582

Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method

PubMed Central

2013-01-01

Background Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. Methods We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Results and conclusions Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting structural states that are significantly different. A detailed analysis on the depth and breadth of the tree suggests that a soft global bias over the progress coordinate enhances sampling and results in higher path diversity. The explicit geometric projection layer that biases the exploration away from over-sampled regions further increases coverage, often improving proximity to the goal by forcing the exploration to find new paths. The reactive temperature scheme is shown effective in increasing path diversity, particularly in difficult structural transitions with known high-energy barriers. PMID:24565158

Exploration of structural stability in deleterious nsSNPs of the XPA gene: A molecular dynamics approach

PubMed Central

NagaSundaram, N; Priya Doss, C George

2011-01-01

Background: Distinguishing the deleterious from the massive number of non-functional nsSNPs that occur within a single genome is a considerable challenge in mutation research. In this approach, we have used the existing in silico methods to explore the mutation-structure-function relationship in the XPAgene. Materials and Methods: We used the Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen), I-Mutant 2.0, and the Protein Analysis THrough Evolutionary Relationships methods to predict the effects of deleterious nsSNPs on protein function and evaluated the impact of mutation on protein stability by Molecular Dynamics simulations. Results: By comparing the scores of all the four in silico methods, nsSNP with an ID rs104894131 at position C108F was predicted to be highly deleterious. We extended our Molecular dynamics approach to gain insight into the impact of this non-synonymous polymorphism on structural changes that may affect the activity of the XPAgene. Conclusion: Based on the in silico methods score, potential energy, root-mean-square deviation, and root-mean-square fluctuation, we predict that deleterious nsSNP at position C108F would play a significant role in causing disease by the XPA gene. Our approach would present the application of in silicotools in understanding the functional variation from the perspective of structure, evolution, and phenotype. PMID:22190868

Discoveries in Down syndrome: moving basic science to clinical care.

PubMed

Kleschevnikov, A M; Belichenko, P V; Salehi, A; Wu, C

2012-01-01

This review describes recent discoveries in neurobiology of Down syndrome (DS) achieved with use of mouse genetic models and provides an overview of experimental approaches aimed at development of pharmacological restoration of cognitive function in people with this developmental disorder. Changes in structure and function of synaptic connections within the hippocampal formation of DS model mice, as well as alterations in innervations of the hippocampus by noradrenergic and cholinergic neuromodulatory systems, provided important clues for potential pharmacological treatments of cognitive disabilities in DS. Possible molecular and cellular mechanisms underlying this genetic disorder have been addressed. We discuss novel mechanisms engaging misprocessing of amyloid precursor protein (App) and other proteins, through their affect on axonal transport and endosomal dysfunction, to "Alzheimer-type" neurodegenerative processes that affect cognition later in life. In conclusion, a number of therapeutic strategies have been defined that may restore cognitive function in mouse models of DS. In the juvenile and young animals, these strategists focus on restoration of synaptic plasticity, rate of adult neurogenesis, and functions of the neuromodulatory subcortical systems. Later in life, the major focus is on recuperation of misprocessed App and related proteins. It is hoped that the identification of an increasing number of potential targets for pharmacotherapy of cognitive deficits in DS will add to the momentum for creating and completing clinical trials. Copyright © 2012 Elsevier B.V. All rights reserved.

PIAS1 interacts with FLASH and enhances its co-activation of c-Myb

PubMed Central

2011-01-01

Background FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator. Results To find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories. Conclusions We conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci. PMID:21338522

Tight Junction Defects in Atopic Dermatitis

PubMed Central

De Benedetto, Anna; Rafaels, Nicholas M.; McGirt, Laura Y.; Ivanov, Andrei I.; Georas, Steve N.; Cheadle, Chris; Berger, Alan E.; Zhang, Kunzhong; Vidyasagar, Sadasivan; Yoshida, Takeshi; Boguniewicz, Mark; Hata, Tissa; Schneider, Lynda C.; Hanifin, Jon M.; Gallo, Richard L.; Novak, Natalija; Weidinger, Stephan; Beaty, Terri H.; Leung, Donald Y.; Barnes, Kathleen C.; Beck, Lisa A.

2010-01-01

Background Atopic dermatitis (AD) is characterized by dry skin and a hyperreactive immune response to allergens, two cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJ) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. Objective We evaluated the expression/function of the TJ protein, claudin-1 in epithelium from AD and nonatopic (NA) subjects and screened two American populations for SNPs in CLDN1. Methods Expression profiles of nonlesional epithelium from extrinsic AD, NA and psoriasis subjects were generated using Illumina’s BeadChips. Dysregulated intercellular proteins were validated by tissue staining and qPCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed using a knockdown approach in primary human keratinocytes (PHK). Twenty seven haplotype-tagging SNPs in CLDN1 were screened in two independent AD populations. Results We observed strikingly reduced expression of the TJ proteins claudin-1 and -23 only in AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with Th2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro, we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging single nucleotide polymorphisms revealed associations with AD in two North American populations. Conclusion Taken together, these data suggest that an impaired epidermal TJ is a novel feature of skin barrier dysfunction and immune dysregulation observed in AD, and that CLDN1 may be a new susceptibility gene in this disease. PMID:21163515

Characterizing Genes with Distinct Methylation Patterns in the Context of Protein-Protein Interaction Network: Application to Human Brain Tissues

PubMed Central

Zhao, Zheng; Li, Shengli; Bai, Jing; Wu, Aiwei; Jiang, Chunjie; Wang, Yuan; Su, Bin; Li, Xia

2013-01-01

Background DNA methylation is an essential epigenetic mechanism involved in transcriptional control. However, how genes with different methylation patterns are assembled in the protein-protein interaction network (PPIN) remains a mystery. Results In the present study, we systematically dissected the characterization of genes with different methylation patterns in the PPIN. A negative association was detected between the methylation levels in the brain tissues and topological centralities. By focusing on two classes of genes with considerably different methylation levels in the brain tissues, namely the low methylated genes (LMGs) and high methylated genes (HMGs), we found that their organizing principles in the PPIN are distinct. The LMGs tend to be the center of the PPIN, and attacking them causes a more deleterious effect on the network integrity. Furthermore, the LMGs express their functions in a modular pattern and substantial differences in functions are observed between the two types of genes. The LMGs are enriched in the basic biological functions, such as binding activity and regulation of transcription. More importantly, cancer genes, especially recessive cancer genes, essential genes, and aging-related genes were all found more often in the LMGs. Additionally, our analysis presented that the intra-classes communications are enhanced, but inter-classes communications are repressed. Finally, a functional complementation was revealed between methylation and miRNA regulation in the human genome. Conclusions We have elucidated the assembling principles of genes with different methylation levels in the context of the PPIN, providing key insights into the complex epigenetic regulation mechanisms. PMID:23776563

Does long-term creatine supplementation impair kidney function in resistance-trained individuals consuming a high-protein diet?

PubMed Central

2013-01-01

Background The aim of this study was to determine the effects of creatine supplementation on kidney function in resistance-trained individuals ingesting a high-protein diet. Methods A randomized, double-blind, placebo-controlled trial was performed. The participants were randomly allocated to receive either creatine (20 g/d for 5 d followed by 5 g/d throughout the trial) or placebo for 12 weeks. All of the participants were engaged in resistance training and consumed a high-protein diet (i.e., ≥ 1.2 g/Kg/d). Subjects were assessed at baseline (Pre) and after 12 weeks (Post). Glomerular filtration rate was measured by 51Cr-EDTA clearance. Additionally, blood samples and a 24-h urine collection were obtained for other kidney function assessments. Results No significant differences were observed for 51Cr-EDTA clearance throughout the trial (Creatine: Pre 101.42 ± 13.11, Post 108.78 ± 14.41 mL/min/1.73m2; Placebo: Pre 103.29 ± 17.64, Post 106.68 ± 16.05 mL/min/1.73m2; group x time interaction: F = 0.21, p = 0.64). Creatinine clearance, serum and urinary urea, electrolytes, proteinuria, and albuminuria remained virtually unchanged. Conclusions A 12-week creatine supplementation protocol did not affect kidney function in resistance-trained healthy individuals consuming a high-protein diet; thus reinforcing the safety of this dietary supplement. Trial registration ClinicalTrials.gov NCT01817673 PMID:23680457

Gene Expression in Human Accessory Lacrimal Glands of Wolfring

PubMed Central

Ubels, John L.; Gipson, Ilene K.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Van Dyken, Rachel E.; Hatton, Mark P.

2012-01-01

Purpose. The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. Methods. Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. Results. Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. Conclusions. The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs. PMID:22956620

Dry eye symptoms are increased in mice deficient in phospholipid transfer protein (PLTP).

PubMed

Setälä, Niko L; Metso, Jari; Jauhiainen, Matti; Sajantila, Antti; Holopainen, Juha M

2011-05-01

In the tear fluid the outermost part facing the tear-air interface is composed of lipids preventing evaporation of the tears. Phospholipid transfer protein (PLTP) mediates phospholipid transfer processes between serum lipoproteins and is also a normal component of human tears. To study whether PLTP plays any functional role in tear fluid we investigated PLTP-deficient mice, applying functional and morphologic analyses under normal housing and experimentally induced dry eye conditions. Aqueous tear fluid production, corneal epithelial morphology, barrier function, and occludin expression were assessed. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression were shown. These pathologic signs were worsened by experimentally induced dry eye both in wild-type and PLTP knock-out mice. Deficiency in the production of tear PLTP in mice is accompanied by corneal epithelial damage, a feature that is typical in human dry eye syndrome (DES). To complement animal experiments we collected tear fluid from human dry eye patients as well as healthy control subjects. Increased tear fluid PLTP activity was observed among DES patients. In conclusion, the presence of PLTP in tear fluid appears to be essential for maintaining a healthy and functional ocular surface. Increased PLTP activity in human tear fluid in DES patients suggests an ocular surface protective role for this lipid transfer protein. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

PubMed Central

Bhattacharyya, Moitrayee; Vishveshwara, Saraswathi

2009-01-01

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials. PMID:19243584

Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

PubMed Central

Skov, Per Stahl; Johnson, Phil E.; Rigby, Neil M.; Przybylski-Nicaise, Laetitia; Bernard, Hervé; Wal, Jean-Michel; Ballmer-Weber, Barbara; Zuidmeer-Jongejan, Laurian; Szépfalusi, Zsolt; Ruinemans-Koerts, Janneke; Jansen, Ad P. H.; Savelkoul, Huub F. J.; Wichers, Harry J.; Mackie, Alan R.; Mills, Clare E. N.; Adel-Patient, Karine

2011-01-01

Background Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins. PMID:21901150

Visualisation and graph-theoretic analysis of a large-scale protein structural interactome

PubMed Central

Bolser, Dan; Dafas, Panos; Harrington, Richard; Park, Jong; Schroeder, Michael

2003-01-01

Background Large-scale protein interaction maps provide a new, global perspective with which to analyse protein function. PSIMAP, the Protein Structural Interactome Map, is a database of all the structurally observed interactions between superfamilies of protein domains with known three-dimensional structure in the PDB. PSIMAP incorporates both functional and evolutionary information into a single network. Results We present a global analysis of PSIMAP using several distinct network measures relating to centrality, interactivity, fault-tolerance, and taxonomic diversity. We found the following results: Centrality: we show that the center and barycenter of PSIMAP do not coincide, and that the superfamilies forming the barycenter relate to very general functions, while those constituting the center relate to enzymatic activity. Interactivity: we identify the P-loop and immunoglobulin superfamilies as the most highly interactive. We successfully use connectivity and cluster index, which characterise the connectivity of a superfamily's neighbourhood, to discover superfamilies of complex I and II. This is particularly significant as the structure of complex I is not yet solved. Taxonomic diversity: we found that highly interactive superfamilies are in general taxonomically very diverse and are thus amongst the oldest. Fault-tolerance: we found that the network is very robust as for the majority of superfamilies removal from the network will not break up the network. Conclusions Overall, we can single out the P-loop containing nucleotide triphosphate hydrolases superfamily as it is the most highly connected and has the highest taxonomic diversity. In addition, this superfamily has the highest interaction rank, is the barycenter of the network (it has the shortest average path to every other superfamily in the network), and is an articulation vertex, whose removal will disconnect the network. More generally, we conclude that the graph-theoretic and taxonomic analysis of PSIMAP is an important step towards the understanding of protein function and could be an important tool for tracing the evolution of life at the molecular level. PMID:14531933

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

Heat Shock70 Protein Genes and Genetic Susceptibility to Apical Periodontitis

PubMed Central

Maheshwari, Kanwal; Silva, Renato M.; Guajardo-Morales, Leticia; Garlet, Gustavo P.; Vieira, Alexandre R.; Letra, Ariadne

2016-01-01

Introduction Heat shock proteins (HSP) protect cells under adverse conditions such as infection, inflammation, and disease. The differential expression of HSPs in human periapical granulomas suggests a potential role for these proteins in periapical lesion development, which may contribute to different clinical outcomes. Therefore, we hypothesize that polymorphisms in HSP genes leading to perturbed gene expression and protein function may contribute to an individual’s susceptibility to periapical lesion development. Methods Subjects with deep carious lesions, with or without periapical lesions (≥ 3 mm) were recruited at the University of Texas School of Dentistry at Houston and at the University of Pittsburgh. Genomic DNA samples of 400 patients were sorted into 2 groups: 183 cases with deep carious lesions and periapical lesions (cases), and 217 cases with deep carious lesions but without periapical lesions (controls). Eight single nucleotide polymorphisms in HSPA4, HSPA6, HSPA1L, HSPA4L and HSPA9 genes were selected for genotyping. Genotypes were generated by endpoint analysis using Taqman chemistry in a real-time polymerase chain reaction assay. Allele and genotype frequencies were compared among cases and controls using chi-square and Fisher Exact tests as implemented in PLINK v.1.07. In silico analysis of SNP function was performed using Polymorphism Phenotyping V2 and MirSNP softwares. Results Overall, SNPs in HSPA1L and HSPA6 showed significant allelic association with cases of deep caries and periapical lesions (P<0.05). We also observed altered transmission of HSPA1L SNP haplotypes (P=0.03). In silico analysis of HSPA1L rs2075800 function showed that this SNP results in a glutamine to lysine substitution at position 602 of the protein and might affect the stability and function of the final protein. Conclusions Variations in HSPA1L and HSPA6 may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing apical periodontitis. PMID:27567034

Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study

PubMed Central

Taghizadeh, Mohammad; Goliaei, Bahram; Madadkar-Sobhani, Armin

2016-01-01

Background Protein flexibility, which has been referred as a dynamic behavior has various roles in proteins’ functions. Furthermore, for some developed tools in bioinformatics, such as protein-protein docking software, considering the protein flexibility, causes a higher degree of accuracy. Through undertaking the present work, we have accomplished the quantification plus analysis of the variations in the human Cyclin Dependent Kinase 2 (hCDK2) protein flexibility without affecting a significant change in its initial environment or the protein per se. Objectives The main goal of the present research was to calculate variations in the flexibility for each residue of the hCDK2, analysis of their flexibility variations through clustering, and to investigate the functional aspects of the residues with high flexibility variations. Materials and Methods Using Gromacs package (version 4.5.4), three independent molecular dynamics (MD) simulations of the hCDK2 protein (PDB ID: 1HCL) was accomplished with no significant changes in their initial environments, structures, or conformations, followed by Root Mean Square Fluctuations (RMSF) calculation of these MD trajectories. The amount of variations in these three curves of RMSF was calculated using two formulas. Results More than 50% of the variation in the flexibility (the distance between the maximum and the minimum amount of the RMSF) was found at the region of Val-154. As well, there are other major flexibility fluctuations in other residues. These residues were mostly positioned in the vicinity of the functional residues. The subsequent works were done, as followed by clustering all hCDK2 residues into four groups considering the amount of their variability with respect to flexibility and their position in the RMSF curves. Conclusions This work has introduced a new class of flexibility aspect of the proteins’ residues. It could also help designing and engineering proteins, with introducing a new dynamic aspect of hCDK2, and accordingly, for the other similar globular proteins. In addition, it could provide a better computational calculation of the protein flexibility, which is, especially important in the comparative studies of the proteins’ flexibility. PMID:28959320

SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

PubMed

Jiang, Junjun; Li, Liyuan; Xie, Mingchao; Fuji, Ryosuke; Liu, Shangfeng; Yin, Xiaobei; Li, Genlin; Wang, Zhao

2015-01-01

Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases

PubMed Central

2014-01-01

Background Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. Results We have developed ngs.plot – a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. Conclusions We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data. PMID:24735413

Consistency of gene starts among Burkholderia genomes

PubMed Central

2011-01-01

Background Evolutionary divergence in the position of the translational start site among orthologous genes can have significant functional impacts. Divergence can alter the translation rate, degradation rate, subcellular location, and function of the encoded proteins. Results Existing Genbank gene maps for Burkholderia genomes suggest that extensive divergence has occurred--53% of ortholog sets based on Genbank gene maps had inconsistent gene start sites. However, most of these inconsistencies appear to be gene-calling errors. Evolutionary divergence was the most plausible explanation for only 17% of the ortholog sets. Correcting probable errors in the Genbank gene maps decreased the percentage of ortholog sets with inconsistent starts by 68%, increased the percentage of ortholog sets with extractable upstream intergenic regions by 32%, increased the sequence similarity of intergenic regions and predicted proteins, and increased the number of proteins with identifiable signal peptides. Conclusions Our findings highlight an emerging problem in comparative genomics: single-digit percent errors in gene predictions can lead to double-digit percentages of inconsistent ortholog sets. The work demonstrates a simple approach to evaluate and improve the quality of gene maps. PMID:21342528

Intracavernous Delivery of a Designed Angiopoietin-1 Variant Rescues Erectile Function by Enhancing Endothelial Regeneration in the Streptozotocin-Induced Diabetic Mouse

PubMed Central

Jin, Hai-Rong; Kim, Woo Jean; Song, Jae Sook; Piao, Shuguang; Choi, Min Ji; Tumurbaatar, Munkhbayar; Shin, Sun Hwa; Yin, Guo Nan; Koh, Gou Young; Ryu, Ji-Kan; Suh, Jun-Kyu

2011-01-01

OBJECTIVE Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. RESEARCH DESIGN AND METHODS Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days −3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. RESULTS Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47phox and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1–treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1–induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. CONCLUSIONS These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction. PMID:21270241

Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

PubMed Central

Ramalho, José S; Anders, Ross; Jaissle, Gesine B; Seeliger, Mathias W; Huxley, Clare; Seabra, Miguel C

2002-01-01

Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases. PMID:12401133

The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

PubMed Central

Winter, Jody A; Christofi, Panayiotis; Morroll, Shaun; Bunting, Karen A

2009-01-01

Background The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å. Results The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions. PMID:19698123

Improving predicted protein loop structure ranking using a Pareto-optimality consensus method

PubMed Central

2010-01-01

Background Accurate protein loop structure models are important to understand functions of many proteins. Identifying the native or near-native models by distinguishing them from the misfolded ones is a critical step in protein loop structure prediction. Results We have developed a Pareto Optimal Consensus (POC) method, which is a consensus model ranking approach to integrate multiple knowledge- or physics-based scoring functions. The procedure of identifying the models of best quality in a model set includes: 1) identifying the models at the Pareto optimal front with respect to a set of scoring functions, and 2) ranking them based on the fuzzy dominance relationship to the rest of the models. We apply the POC method to a large number of decoy sets for loops of 4- to 12-residue in length using a functional space composed of several carefully-selected scoring functions: Rosetta, DOPE, DDFIRE, OPLS-AA, and a triplet backbone dihedral potential developed in our lab. Our computational results show that the sets of Pareto-optimal decoys, which are typically composed of ~20% or less of the overall decoys in a set, have a good coverage of the best or near-best decoys in more than 99% of the loop targets. Compared to the individual scoring function yielding best selection accuracy in the decoy sets, the POC method yields 23%, 37%, and 64% less false positives in distinguishing the native conformation, indentifying a near-native model (RMSD < 0.5A from the native) as top-ranked, and selecting at least one near-native model in the top-5-ranked models, respectively. Similar effectiveness of the POC method is also found in the decoy sets from membrane protein loops. Furthermore, the POC method outperforms the other popularly-used consensus strategies in model ranking, such as rank-by-number, rank-by-rank, rank-by-vote, and regression-based methods. Conclusions By integrating multiple knowledge- and physics-based scoring functions based on Pareto optimality and fuzzy dominance, the POC method is effective in distinguishing the best loop models from the other ones within a loop model set. PMID:20642859

Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

PubMed Central

Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

2014-01-01

Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

Biophysical and structural considerations for protein sequence evolution

PubMed Central

2011-01-01

Background Protein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field. Results Here we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS < 1 and gamma-distributed rates across sites. Conclusions Simple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model. PMID:22171550

Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis

PubMed Central

2012-01-01

Background GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. Results In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes. Conclusions Our current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study. PMID:22793791

In silico prediction of protein-protein interactions in human macrophages

PubMed Central

2014-01-01

Background Protein-protein interaction (PPI) network analyses are highly valuable in deciphering and understanding the intricate organisation of cellular functions. Nevertheless, the majority of available protein-protein interaction networks are context-less, i.e. without any reference to the spatial, temporal or physiological conditions in which the interactions may occur. In this work, we are proposing a protocol to infer the most likely protein-protein interaction (PPI) network in human macrophages. Results We integrated the PPI dataset from the Agile Protein Interaction DataAnalyzer (APID) with different meta-data to infer a contextualized macrophage-specific interactome using a combination of statistical methods. The obtained interactome is enriched in experimentally verified interactions and in proteins involved in macrophage-related biological processes (i.e. immune response activation, regulation of apoptosis). As a case study, we used the contextualized interactome to highlight the cellular processes induced upon Mycobacterium tuberculosis infection. Conclusion Our work confirms that contextualizing interactomes improves the biological significance of bioinformatic analyses. More specifically, studying such inferred network rather than focusing at the gene expression level only, is informative on the processes involved in the host response. Indeed, important immune features such as apoptosis are solely highlighted when the spotlight is on the protein interaction level. PMID:24636261

Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy

PubMed Central

Hershberger, Ray E.; Pinto, Jose Renato; Parks, Sharie B.; Kushner, Jessica D.; Li, Duanxiang; Ludwigsen, Susan; Cowan, Jason; Morales, Ana; Parvatiyar, Michelle S.; Potter, James D.

2009-01-01

Background A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. Methods and Results We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified six TNNT2 protein-altering variants in nine probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the nine probands had DCM without a family history, and five had familial DCM. Only one mutation (Lys210del) could be attributed as definitively causative from prior reports. Four of the five missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree and molecular genetic data these five mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca2+ sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease-causing. Conclusions We conclude that the combination of clinical, pedigree, molecular genetic and functional data strengthen the interpretation of TNNT2 mutations in DCM. PMID:20031601

Design and engineering of water-soluble light-harvesting protein maquettes

DOE PAGES

Kodali, Goutham; Mancini, Joshua A.; Solomon, Lee A.; ...

2017-01-01

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistrymore » of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. In conclusion, this partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.« less

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

PubMed Central

2014-01-01

Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238

Design and engineering of water-soluble light-harvesting protein maquettes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kodali, Goutham; Mancini, Joshua A.; Solomon, Lee A.

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistrymore » of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. In conclusion, this partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.« less

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

PubMed

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

2012-12-01

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

DOE PAGES

Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; ...

2014-12-08

Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1more » acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.« less

RAPSearch: a fast protein similarity search tool for short reads

PubMed Central

2011-01-01

Background Next Generation Sequencing (NGS) is producing enormous corpuses of short DNA reads, affecting emerging fields like metagenomics. Protein similarity search--a key step to achieve annotation of protein-coding genes in these short reads, and identification of their biological functions--faces daunting challenges because of the very sizes of the short read datasets. Results We developed a fast protein similarity search tool RAPSearch that utilizes a reduced amino acid alphabet and suffix array to detect seeds of flexible length. For short reads (translated in 6 frames) we tested, RAPSearch achieved ~20-90 times speedup as compared to BLASTX. RAPSearch missed only a small fraction (~1.3-3.2%) of BLASTX similarity hits, but it also discovered additional homologous proteins (~0.3-2.1%) that BLASTX missed. By contrast, BLAT, a tool that is even slightly faster than RAPSearch, had significant loss of sensitivity as compared to RAPSearch and BLAST. Conclusions RAPSearch is implemented as open-source software and is accessible at http://omics.informatics.indiana.edu/mg/RAPSearch. It enables faster protein similarity search. The application of RAPSearch in metageomics has also been demonstrated. PMID:21575167

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies.

PubMed

Zeilinger, Katrin; Schreiter, Thomas; Darnell, Malin; Söderdahl, Therese; Lübberstedt, Marc; Dillner, Birgitta; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B

2011-05-01

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.

Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

PubMed Central

Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

2012-01-01

Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

Selective Individual Primary Cell Capture Using Locally Bio-Functionalized Micropores

PubMed Central

Liu, Jie; Bombera, Radoslaw; Leroy, Loïc; Roupioz, Yoann; Baganizi, Dieudonné R.; Marche, Patrice N.; Haguet, Vincent; Mailley, Pascal; Livache, Thierry

2013-01-01

Background Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. Methodology/Principal Findings We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. Conclusions/Significance The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures. PMID:23469221

Identification of proteins involved in the pancreatic exocrine by exogenous ghrelin administration in Sprague-Dawley rats.

PubMed

Lee, Kyung-Hoon; Wang, Tao; Jin, Yong-Cheng; Lee, Sang-Bum; Oh, Jin-Ju; Hwang, Jin-Hee; Lim, Ji-Na; Lee, Jae-Sung; Lee, Hong-Gu

2014-01-01

The aims of study were to investigate the effects of intraperitoneal (i.p.) infusion of ghrelin on pancreatic α-amylase outputs and the responses of pancreatic proteins to ghrelin that may relate to the pancreatic exocrine. Six male Sprague-Dawley rats (300 g) were randomly divided into two groups, a control group (C, n = 3) and a treatment group (T, 10.0μg/kg BW, n = 3). Blood samples were collected from rat caudal vein once time after one hour injection. The concentrations of plasma ghrelin, cholecystokinin (CCK) and alfa-amylase activity were evaluated by enzyme immunoassay (EIA) kit. Two-dimensional gel electrophoresis (2-DE) analysis was conducted to separate the proteins in pancreas tissue. Results showed that the i.p. infusion of ghrelin at doses of 10.0 μg/kg body weight (BW) increased the plasma ghrelin concentrations (p = 0.07) and elevated the plasma CCK level significantly (p < 0.05). Although there was no statistically significant, the α-amylase activity tended to increase. The proteomics analysis indicated that some pancreatic proteins with various functions were up- or down- regulated compared with control group. In conclusion, ghrelin may have role in the pancreatic exocrine, but the signaling pathway was still not clear. Therefore, much more functional studies focus on these found proteins are needed in the near future.

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

PubMed

Yang, Tai-Yun; Chiang, Nien-Yi; Tseng, Wen-Yi; Pan, Hsiao-Lin; Peng, Yen-Ming; Shen, Jiann-Jong; Wu, Kuo-An; Kuo, Ming-Ling; Chang, Gin-Wen; Lin, Hsi-Hsien

2015-05-01

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of Protein Flexibility in Ion Permeation: A Case Study in Gramicidin A

PubMed Central

Baştuğ, Turgut; Gray-Weale, Angus; Patra, Swarna M.; Kuyucak, Serdar

2006-01-01

Proteins have a flexible structure, and their atoms exhibit considerable fluctuations under normal operating conditions. However, apart from some enzyme reactions involving ligand binding, our understanding of the role of flexibility in protein function remains mostly incomplete. Here we investigate this question in the realm of membrane proteins that form ion channels. Specifically, we consider ion permeation in the gramicidin A channel, and study how the energetics of ion conduction changes as the channel structure is progressively changed from completely flexible to a fixed one. For each channel structure, the potential of mean force for a permeating potassium ion is determined from molecular dynamics (MD) simulations. Using the same molecular dynamics data for completely flexible gramicidin A, we also calculate the average densities and fluctuations of the peptide atoms and investigate the correlations between these fluctuations and the motion of a permeating ion. Our results show conclusively that peptide flexibility plays an important role in ion permeation in the gramicidin A channel, thus providing another reason—besides the well-known problem with the description of single file pore water—why this channel cannot be modeled using continuum electrostatics with a fixed structure. The new method developed here for studying the role of protein flexibility on its function clarifies the contributions of the fluctuations to energy and entropy, and places limits on the level of detail required in a coarse-grained model. PMID:16415054

Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

PubMed

Salmon, Magali S; Bayer, Emmanuelle M F

2012-01-01

In plants, the intercellular communication through the membranous channels called plasmodesmata (PD; singular plasmodesma) plays pivotal roles in the orchestration of development, defence responses, and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialized domains of the endoplasmic reticulum (ER) and the plasma membrane (PM). PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalization, or screening of random cDNAs, only few PD proteins had been conclusively identified and characterized. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on "free" PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic-based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD-associated proteins.

Structural and functional effects of nucleotide variation on the human TB drug metabolizing enzyme arylamine N-acetyltransferase 1.

PubMed

Cloete, Ruben; Akurugu, Wisdom A; Werely, Cedric J; van Helden, Paul D; Christoffels, Alan

2017-08-01

The human arylamine N-acetyltransferase 1 (NAT1) enzyme plays a vital role in determining the duration of action of amine-containing drugs such as para-aminobenzoic acid (PABA) by influencing the balance between detoxification and metabolic activation of these drugs. Recently, four novel single nucleotide polymorphisms (SNPs) were identified within a South African mixed ancestry population. Modeling the effects of these SNPs within the structural protein was done to assess possible structure and function changes in the enzyme. The use of molecular dynamics simulations and stability predictions indicated less thermodynamically stable protein structures containing E264K and V231G, while the N245I change showed a stabilizing effect. Coincidently the N245I change displayed a similar free energy landscape profile to the known R64W amino acid substitution (slow acetylator), while the R242M displayed a similar profile to the published variant, I263V (proposed fast acetylator), and the wild type protein structure. Similarly, principal component analysis indicated that two amino acid substitutions (E264K and V231G) occupied less conformational clusters of folded states as compared to the WT and were found to be destabilizing (may affect protein function). However, two of the four novel SNPs that result in amino acid changes: (V231G and N245I) were predicted by both SIFT and POLYPHEN-2 algorithms to affect NAT1 protein function, while two other SNPs that result in R242M and E264K substitutions showed contradictory results based on SIFT and POLYPHEN-2 analysis. In conclusion, the structural methods were able to verify that two non-synonymous substitutions (E264K and V231G) can destabilize the protein structure, and are in agreement with mCSM predictions, and should therefore be experimentally tested for NAT1 activity. These findings could inform a strategy of incorporating genotypic data (i.e., functional SNP alleles) with phenotypic information (slow or fast acetylator) to better prescribe effective treatment using drugs metabolized by NAT1. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A protein domain-centric approach for the comparative analysis of human and yeast phenotypically relevant mutations

PubMed Central

2013-01-01

Background The body of disease mutations with known phenotypic relevance continues to increase and is expected to do so even faster with the advent of new experimental techniques such as whole-genome sequencing coupled with disease association studies. However, genomic association studies are limited by the molecular complexity of the phenotype being studied and the population size needed to have adequate statistical power. One way to circumvent this problem, which is critical for the study of rare diseases, is to study the molecular patterns emerging from functional studies of existing disease mutations. Current gene-centric analyses to study mutations in coding regions are limited by their inability to account for the functional modularity of the protein. Previous studies of the functional patterns of known human disease mutations have shown a significant tendency to cluster at protein domain positions, namely position-based domain hotspots of disease mutations. However, the limited number of known disease mutations remains the main factor hindering the advancement of mutation studies at a functional level. In this paper, we address this problem by incorporating mutations known to be disruptive of phenotypes in other species. Focusing on two evolutionarily distant organisms, human and yeast, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level. Results The results of this analysis reveal that phenotypic mutations from yeast cluster at specific positions on protein domains, a characteristic previously revealed to be displayed by human disease mutations. We found over one hundred domain hotspots in yeast with approximately 50% in the exact same domain position as known human disease mutations. Conclusions We describe an analysis using protein domains as a framework for transferring functional information by studying domain hotspots in human and yeast and relating phenotypic changes in yeast to diseases in human. This first-of-a-kind study of phenotypically relevant yeast mutations in relation to human disease mutations demonstrates the utility of a multi-species analysis for advancing the understanding of the relationship between genetic mutations and phenotypic changes at the organismal level. PMID:23819456

Protein structure and evolution: are they constrained globally by a principle derived from information theory?

PubMed

Hatton, Leslie; Warr, Gregory

2015-01-01

That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

PubMed Central

Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan MHAI; Mondol, Sobuj; Ahmed, C M Sabbir

2014-01-01

Purpose Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Conclusion Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to develop and discover more effective and specific therapeutic agents against S. sanguinis. PMID:25473301

GOGrapher: A Python library for GO graph representation and analysis

PubMed Central

Muller, Brian; Richards, Adam J; Jin, Bo; Lu, Xinghua

2009-01-01

Background The Gene Ontology is the most commonly used controlled vocabulary for annotating proteins. The concepts in the ontology are organized as a directed acyclic graph, in which a node corresponds to a biological concept and a directed edge denotes the parent-child semantic relationship between a pair of terms. A large number of protein annotations further create links between proteins and their functional annotations, reflecting the contemporary knowledge about proteins and their functional relationships. This leads to a complex graph consisting of interleaved biological concepts and their associated proteins. What is needed is a simple, open source library that provides tools to not only create and view the Gene Ontology graph, but to analyze and manipulate it as well. Here we describe the development and use of GOGrapher, a Python library that can be used for the creation, analysis, manipulation, and visualization of Gene Ontology related graphs. Findings An object-oriented approach was adopted to organize the hierarchy of the graphs types and associated classes. An Application Programming Interface is provided through which different types of graphs can be pragmatically created, manipulated, and visualized. GOGrapher has been successfully utilized in multiple research projects, e.g., a graph-based multi-label text classifier for protein annotation. Conclusion The GOGrapher project provides a reusable programming library designed for the manipulation and analysis of Gene Ontology graphs. The library is freely available for the scientific community to use and improve. PMID:19583843

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

PubMed Central

2014-01-01

Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

Proteomic profile of dormant Trichophyton Rubrum conidia

PubMed Central

Leng, Wenchuan; Liu, Tao; Li, Rui; Yang, Jian; Wei, Candong; Zhang, Wenliang; Jin, Qi

2008-01-01

Background Trichophyton rubrum is the most common dermatophyte causing fungal skin infections in humans. Asexual sporulation is an important means of propagation for T. rubrum, and conidia produced by this way are thought to be the primary cause of human infections. Despite their importance in pathogenesis, the conidia of T. rubrum remain understudied. We intend to intensively investigate the proteome of dormant T. rubrum conidia to characterize its molecular and cellular features and to enhance the development of novel therapeutic strategies. Results The proteome of T. rubrum conidia was analyzed by combining shotgun proteomics with sample prefractionation and multiple enzyme digestion. In total, 1026 proteins were identified. All identified proteins were compared to those in the NCBI non-redundant protein database, the eukaryotic orthologous groups database, and the gene ontology database to obtain functional annotation information. Functional classification revealed that the identified proteins covered nearly all major biological processes. Some proteins were spore specific and related to the survival and dispersal of T. rubrum conidia, and many proteins were important to conidial germination and response to environmental conditions. Conclusion Our results suggest that the proteome of T. rubrum conidia is considerably complex, and that the maintenance of conidial dormancy is an intricate and elaborate process. This data set provides the first global framework for the dormant T. rubrum conidia proteome and is a stepping stone on the way to further study of the molecular mechanisms of T. rubrum conidial germination and the maintenance of conidial dormancy. PMID:18578874

Characterization of the duck enteritis virus UL55 protein

PubMed Central

2011-01-01

Background Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function. Results The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells. Conclusions In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene. PMID:21609474

Transcriptome Analysis of Flower Sex Differentiation in Jatropha curcas L. Using RNA Sequencing

PubMed Central

Xu, Gang; Huang, Jian; Yang, Yong; Yao, Yin-an

2016-01-01

Background Jatropha curcas is thought to be a promising biofuel material, but its yield is restricted by a low ratio of instaminate / staminate flowers (1/10-1/30). Furthermore, valuable information about flower sex differentiation in this plant is scarce. To explore the mechanism of this process in J. curcas, transcriptome profiling of flower development was carried out, and certain genes related with sex differentiation were obtained through digital gene expression analysis of flower buds from different phases of floral development. Results After Illumina sequencing and clustering, 57,962 unigenes were identified. A total of 47,423 unigenes were annotated, with 85 being related to carpel and stamen differentiation, 126 involved in carpel and stamen development, and 592 functioning in the later development stage for the maturation of staminate or instaminate flowers. Annotation of these genes provided comprehensive information regarding the sex differentiation of flowers, including the signaling system, hormone biosynthesis and regulation, transcription regulation and ubiquitin-mediated proteolysis. A further expression pattern analysis of 15 sex-related genes using quantitative real-time PCR revealed that gibberellin-regulated protein 4-like protein and AMP-activated protein kinase are associated with stamen differentiation, whereas auxin response factor 6-like protein, AGAMOUS-like 20 protein, CLAVATA1, RING-H2 finger protein ATL3J, auxin-induced protein 22D, and r2r3-myb transcription factor contribute to embryo sac development in the instaminate flower. Cytokinin oxidase, Unigene28, auxin repressed-like protein ARP1, gibberellin receptor protein GID1 and auxin-induced protein X10A are involved in both stages mentioned above. In addition to its function in the differentiation and development of the stamens, the gibberellin signaling pathway also functions in embryo sac development for the instaminate flower. The auxin signaling pathway also participates in both stamen development and embryo sac development. Conclusions Our transcriptome data provide a comprehensive gene expression profile for flower sex differentiation in Jatropha curcas, as well as new clues and information for further study in this field. PMID:26848843

Identification of a G‐Protein Subunit‐α11 Gain‐of‐Function Mutation, Val340Met, in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2)

PubMed Central

Piret, Sian E; Gorvin, Caroline M; Pagnamenta, Alistair T; Howles, Sarah A; Cranston, Treena; Rust, Nigel; Nesbit, M Andrew; Glaser, Ben; Taylor, Jenny C; Buchs, Andreas E; Hannan, Fadil M

2016-01-01

ABSTRACT Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα11). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC50) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C‐terminal region for G‐protein function and CaSR signal transduction. © 2016 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR). PMID:26818911

Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family

PubMed Central

Jung, Du-Kyo; Lee, Youra; Park, Sung Goo; Park, Byoung Chul; Kim, Ghyung-Hwa; Rhee, Sangkee

2006-01-01

The ureide pathway, which produces ureides from uric acid, is an essential purine catabolic process for storing and transporting the nitrogen fixed in leguminous plants and some bacteria. PucM from Bacillus subtilis was recently characterized and found to catalyze the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step. PucM has 121 amino acid residues and shows high sequence similarity to the functionally unrelated protein transthyretin (TTR), a thyroid hormone-binding protein. Therefore, PucM belongs to the TTR-related proteins (TRP) family. The crystal structures of PucM at 2.0 Å and its complexes with the substrate analogs 8-azaxanthine and 5,6-diaminouracil reveal that even with their overall structure similarity, homotetrameric PucM and TTR are completely different, both in their electrostatic potential and in the size of the active sites located at the dimeric interface. Nevertheless, the absolutely conserved residues across the TRP family, including His-14, Arg-49, His-105, and the C-terminal Tyr-118–Arg-119–Gly-120–Ser-121, indeed form the active site of PucM. Based on the results of site-directed mutagenesis of these residues, we propose a possible mechanism for HIU hydrolysis. The PucM structure determined for the TRP family leads to the conclusion that diverse members of the TRP family would function similarly to PucM as HIU hydrolase. PMID:16782815

MINE: Module Identification in Networks

PubMed Central

2011-01-01

Background Graphical models of network associations are useful for both visualizing and integrating multiple types of association data. Identifying modules, or groups of functionally related gene products, is an important challenge in analyzing biological networks. However, existing tools to identify modules are insufficient when applied to dense networks of experimentally derived interaction data. To address this problem, we have developed an agglomerative clustering method that is able to identify highly modular sets of gene products within highly interconnected molecular interaction networks. Results MINE outperforms MCODE, CFinder, NEMO, SPICi, and MCL in identifying non-exclusive, high modularity clusters when applied to the C. elegans protein-protein interaction network. The algorithm generally achieves superior geometric accuracy and modularity for annotated functional categories. In comparison with the most closely related algorithm, MCODE, the top clusters identified by MINE are consistently of higher density and MINE is less likely to designate overlapping modules as a single unit. MINE offers a high level of granularity with a small number of adjustable parameters, enabling users to fine-tune cluster results for input networks with differing topological properties. Conclusions MINE was created in response to the challenge of discovering high quality modules of gene products within highly interconnected biological networks. The algorithm allows a high degree of flexibility and user-customisation of results with few adjustable parameters. MINE outperforms several popular clustering algorithms in identifying modules with high modularity and obtains good overall recall and precision of functional annotations in protein-protein interaction networks from both S. cerevisiae and C. elegans. PMID:21605434

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

PubMed Central

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-01-01

AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

PubMed

Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

2016-11-01

The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Computational Identification and Functional Predictions of Long Noncoding RNA in Zea mays

PubMed Central

Boerner, Susan; McGinnis, Karen M.

2012-01-01

Background Computational analysis of cDNA sequences from multiple organisms suggests that a large portion of transcribed DNA does not code for a functional protein. In mammals, noncoding transcription is abundant, and often results in functional RNA molecules that do not appear to encode proteins. Many long noncoding RNAs (lncRNAs) appear to have epigenetic regulatory function in humans, including HOTAIR and XIST. While epigenetic gene regulation is clearly an essential mechanism in plants, relatively little is known about the presence or function of lncRNAs in plants. Methodology/Principal Findings To explore the connection between lncRNA and epigenetic regulation of gene expression in plants, a computational pipeline using the programming language Python has been developed and applied to maize full length cDNA sequences to identify, classify, and localize potential lncRNAs. The pipeline was used in parallel with an SVM tool for identifying ncRNAs to identify the maximal number of ncRNAs in the dataset. Although the available library of sequences was small and potentially biased toward protein coding transcripts, 15% of the sequences were predicted to be noncoding. Approximately 60% of these sequences appear to act as precursors for small RNA molecules and may function to regulate gene expression via a small RNA dependent mechanism. ncRNAs were predicted to originate from both genic and intergenic loci. Of the lncRNAs that originated from genic loci, ∼20% were antisense to the host gene loci. Conclusions/Significance Consistent with similar studies in other organisms, noncoding transcription appears to be widespread in the maize genome. Computational predictions indicate that maize lncRNAs may function to regulate expression of other genes through multiple RNA mediated mechanisms. PMID:22916204

A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

PubMed Central

Irshad, Muhammad; Canut, Hervé; Borderies, Gisèle; Pont-Lezica, Rafael; Jamet, Elisabeth

2008-01-01

Background Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time. Results Two developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function. Conclusion This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins. PMID:18796151

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

PubMed

Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

2018-01-05

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

Autoinhibition and signaling by the switch II motif in the G-protein chaperone of a radical B12 enzyme.

PubMed

Lofgren, Michael; Koutmos, Markos; Banerjee, Ruma

2013-10-25

MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.

Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life.

PubMed

Lopes, Luísa V; Marvin-Guy, Laure F; Fuerholz, Andreas; Affolter, Michael; Ramadan, Ziad; Kussmann, Martin; Fay, Laurent B; Bergonzelli, Gabriela E

2008-04-30

Early life stress as neonatal maternal deprivation (MD) predisposes rats to alter gut functions in response to acute psychological stressors in adulthood, mimicking features of irritable bowel syndrome (IBS). We applied proteomics to investigate whether MD permanently changes the protein profile of the external colonic neuromuscular layer that may condition the molecular response to an acute stressor later in life. Male rat pups were separated 3 h/day from their mothers during the perinatal period and further submitted to water avoidance (WA) stress during adulthood. Proteins were extracted from the myenteric plexus-longitudinal muscle of control (C), WA and MD+WA rat colon, separated on 2D gels, and identified by mass spectrometry. MD amplified the WA-induced protein changes involved in muscle contractile function, suggesting that stress accumulation along life imbalances the muscle tone towards hypercontractility. Our results also propose a stress dependent regulation of gluconeogenesis. Secretogranin II - the secretoneurin precursor - was induced by MD. The presence of secretoneurin in myenteric ganglia may partially explain the stress-mediated modulation of gastrointestinal motility and/or mucosal inflammation previously described in MD rats. In conclusion, our findings suggest that neonatal stress alters the responses to acute stress in adulthood in intestinal smooth muscle and enteric neurons.

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency

PubMed Central

2013-01-01

Background The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. Methods We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Results Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. Conclusions A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD. PMID:24289245

Analysis of the Human Prostate-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling Identifies TMEM79 and ACOXL as Two Putative, Diagnostic Markers in Prostate Cancer

PubMed Central

O'Hurley, Gillian; Busch, Christer; Fagerberg, Linn; Hallström, Björn M.; Stadler, Charlotte; Tolf, Anna; Lundberg, Emma; Schwenk, Jochen M.; Jirström, Karin; Bjartell, Anders; Gallagher, William M.; Uhlén, Mathias; Pontén, Fredrik

2015-01-01

To better understand prostate function and disease, it is important to define and explore the molecular constituents that signify the prostate gland. The aim of this study was to define the prostate specific transcriptome and proteome, in comparison to 26 other human tissues. Deep sequencing of mRNA (RNA-seq) and immunohistochemistry-based protein profiling were combined to identify prostate specific gene expression patterns and to explore tissue biomarkers for potential clinical use in prostate cancer diagnostics. We identified 203 genes with elevated expression in the prostate, 22 of which showed more than five-fold higher expression levels compared to all other tissue types. In addition to previously well-known proteins we identified two poorly characterized proteins, TMEM79 and ACOXL, with potential to differentiate between benign and cancerous prostatic glands in tissue biopsies. In conclusion, we have applied a genome-wide analysis to identify the prostate specific proteome using transcriptomics and antibody-based protein profiling to identify genes with elevated expression in the prostate. Our data provides a starting point for further functional studies to explore the molecular repertoire of normal and diseased prostate including potential prostate cancer markers such as TMEM79 and ACOXL. PMID:26237329

TRPV3 Channel in Keratinocytes in Scars with Post-Burn Pruritus

PubMed Central

Park, Chun Wook; Kim, Hyun Ji; Choi, Yong Won; Chung, Bo Young; Woo, So-Youn; Song, Dong-Keun; Kim, Hye One

2017-01-01

Post-burn pruritus is a common and distressing sequela of burn scars. Empirical antipruritic treatments usually fail to have a satisfactory outcome because of their limited selectivity and possible side effects. Therefore, novel drug targets need to be identified. Here, we aimed to investigate the possible role of protease-activated receptor 2 (PAR2) and transient receptor potential vanniloid 3 (TRPV3), along with the relation of TRPV3 to thymic stromal lymphopoietin (TSLP). Specimens from normal (unscarred) or burn-scarred (with or without pruritus) tissue were obtained from burn patients for this study. In each sample, the keratinocytes were isolated and cultured, and the intracellular Ca2+ level at the time of stimulation of each factor was quantified and the interaction was screened. PAR2 function was reduced by antagonism of TRPV3. Inhibiting protein kinase A (PKA) and protein kinase C (PKC) reduced TRPV3 function. TSLP mRNA and protein, and TSLPR protein expressions, increased in scars with post-burn pruritus, compared to scars without it or to normal tissues. In addition, TRPV1 or TRPV3 activation induced increased TSLP expression. Conclusively, TRPV3 may contribute to pruritus in burn scars through TSLP, and can be considered a potential therapeutic target for post-burn pruritus. PMID:29140280

Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands.

PubMed

Fang, Dongdong; Hu, Shen; Liu, Younan; Quan, Vu-Hung; Seuntjens, Jan; Tran, Simon D

2015-11-03

In separate studies, an extract of soluble intracellular contents from whole bone marrow cells, named "Bone Marrow (BM) Soup", was reported to either improve cardiac or salivary functions post-myocardial infarction or irradiation (IR), respectively. However, the active components in BM Soup are unknown. To demonstrate that proteins were the active ingredients, we devised a method using proteinase K followed by heating to deactivate proteins and for safe injections into mice. BM Soup and "deactivated BM Soup" were injected into mice that had their salivary glands injured with 15Gy IR. Control mice received either injections of saline or were not IR. Results at week 8 post-IR showed the 'deactivated BM Soup' was no better than injections of saline, while injections of native BM Soup restored saliva flow, protected salivary cells and blood vessels from IR-damage. Protein arrays detected several angiogenesis-related factors (CD26, FGF, HGF, MMP-8, MMP-9, OPN, PF4, SDF-1) and cytokines (IL-1ra, IL-16) in BM Soup. In conclusion, the native proteins (but not the nucleic acids, lipids or carbohydrates) were the therapeutic ingredients in BM Soup for functional salivary restoration following IR. This molecular therapy approach has clinical potential because it is theoretically less tumorigenic and immunogenic than cell therapies.

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

PubMed Central

Cavalieri, Vincenzo; Melfi, Raffaella; Spinelli, Giovanni

2013-01-01

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts. PMID:24086165

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

PubMed Central

Dreyfus, David H.

2009-01-01

Background The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the “RAG transposon”. Methodology/Principal Findings Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a “RAG transposon.” A subsequent “arms race” between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex). Conclusions/Significance A “co-regulatory” model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the “RAG-transposon” hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination. PMID:19492059

Reconstruction of the experimentally supported human protein interactome: what can we learn?

PubMed Central

2013-01-01

Background Understanding the topology and dynamics of the human protein-protein interaction (PPI) network will significantly contribute to biomedical research, therefore its systematic reconstruction is required. Several meta-databases integrate source PPI datasets, but the protein node sets of their networks vary depending on the PPI data combined. Due to this inherent heterogeneity, the way in which the human PPI network expands via multiple dataset integration has not been comprehensively analyzed. We aim at assembling the human interactome in a global structured way and exploring it to gain insights of biological relevance. Results First, we defined the UniProtKB manually reviewed human “complete” proteome as the reference protein-node set and then we mined five major source PPI datasets for direct PPIs exclusively between the reference proteins. We updated the protein and publication identifiers and normalized all PPIs to the UniProt identifier level. The reconstructed interactome covers approximately 60% of the human proteome and has a scale-free structure. No apparent differentiating gene functional classification characteristics were identified for the unrepresented proteins. The source dataset integration augments the network mainly in PPIs. Polyubiquitin emerged as the highest-degree node, but the inclusion of most of its identified PPIs may be reconsidered. The high number (>300) of connections of the subsequent fifteen proteins correlates well with their essential biological role. According to the power-law network structure, the unrepresented proteins should mainly have up to four connections with equally poorly-connected interactors. Conclusions Reconstructing the human interactome based on the a priori definition of the protein nodes enabled us to identify the currently included part of the human “complete” proteome, and discuss the role of the proteins within the network topology with respect to their function. As the network expansion has to comply with the scale-free theory, we suggest that the core of the human interactome has essentially emerged. Thus, it could be employed in systems biology and biomedical research, despite the considerable number of currently unrepresented proteins. The latter are probably involved in specialized physiological conditions, justifying the scarcity of related PPI information, and their identification can assist in designing relevant functional experiments and targeted text mining algorithms. PMID:24088582

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae

PubMed Central

Meng, Shaowu; Brown, Douglas E; Ebbole, Daniel J; Torto-Alalibo, Trudy; Oh, Yeon Yee; Deng, Jixin; Mitchell, Thomas K; Dean, Ralph A

2009-01-01

Background Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 . However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. Methods A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. Results In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site . Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website . The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. Conclusion Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae. PMID:19278556

Reduced functionality of PSE-like chicken breast meat batter resulting from alterations in protein conformation.

PubMed

Li, K; Zhao, Y Y; Kang, Z L; Wang, P; Han, M Y; Xu, X L; Zhou, G H

2015-01-01

The objectives of this study were to evaluate protein thermal stability, water-protein interaction, microstructure, and protein conformation between PSE-like and normal chicken breast meat batters. Sixty pale, soft, and exudative (PSE)-like (L*>53, pH24 h<5.7) and 60 normal (46

Protein-protein docking using region-based 3D Zernike descriptors

PubMed Central

2009-01-01

Background Protein-protein interactions are a pivotal component of many biological processes and mediate a variety of functions. Knowing the tertiary structure of a protein complex is therefore essential for understanding the interaction mechanism. However, experimental techniques to solve the structure of the complex are often found to be difficult. To this end, computational protein-protein docking approaches can provide a useful alternative to address this issue. Prediction of docking conformations relies on methods that effectively capture shape features of the participating proteins while giving due consideration to conformational changes that may occur. Results We present a novel protein docking algorithm based on the use of 3D Zernike descriptors as regional features of molecular shape. The key motivation of using these descriptors is their invariance to transformation, in addition to a compact representation of local surface shape characteristics. Docking decoys are generated using geometric hashing, which are then ranked by a scoring function that incorporates a buried surface area and a novel geometric complementarity term based on normals associated with the 3D Zernike shape description. Our docking algorithm was tested on both bound and unbound cases in the ZDOCK benchmark 2.0 dataset. In 74% of the bound docking predictions, our method was able to find a near-native solution (interface C-αRMSD ≤ 2.5 Å) within the top 1000 ranks. For unbound docking, among the 60 complexes for which our algorithm returned at least one hit, 60% of the cases were ranked within the top 2000. Comparison with existing shape-based docking algorithms shows that our method has a better performance than the others in unbound docking while remaining competitive for bound docking cases. Conclusion We show for the first time that the 3D Zernike descriptors are adept in capturing shape complementarity at the protein-protein interface and useful for protein docking prediction. Rigorous benchmark studies show that our docking approach has a superior performance compared to existing methods. PMID:20003235

Re-visiting protein-centric two-tier classification of existing DNA-protein complexes

PubMed Central

2012-01-01

Background Precise DNA-protein interactions play most important and vital role in maintaining the normal physiological functioning of the cell, as it controls many high fidelity cellular processes. Detailed study of the nature of these interactions has paved the way for understanding the mechanisms behind the biological processes in which they are involved. Earlier in 2000, a systematic classification of DNA-protein complexes based on the structural analysis of the proteins was proposed at two tiers, namely groups and families. With the advancement in the number and resolution of structures of DNA-protein complexes deposited in the Protein Data Bank, it is important to revisit the existing classification. Results On the basis of the sequence analysis of DNA binding proteins, we have built upon the protein centric, two-tier classification of DNA-protein complexes by adding new members to existing families and making new families and groups. While classifying the new complexes, we also realised the emergence of new groups and families. The new group observed was where β-propeller was seen to interact with DNA. There were 34 SCOP folds which were observed to be present in the complexes of both old and new classifications, whereas 28 folds are present exclusively in the new complexes. Some new families noticed were NarL transcription factor, Z-α DNA binding proteins, Forkhead transcription factor, AP2 protein, Methyl CpG binding protein etc. Conclusions Our results suggest that with the increasing number of availability of DNA-protein complexes in Protein Data Bank, the number of families in the classification increased by approximately three fold. The folds present exclusively in newly classified complexes is suggestive of inclusion of proteins with new function in new classification, the most populated of which are the folds responsible for DNA damage repair. The proposed re-visited classification can be used to perform genome-wide surveys in the genomes of interest for the presence of DNA-binding proteins. Further analysis of these complexes can aid in developing algorithms for identifying DNA-binding proteins and their family members from mere sequence information. PMID:22800292

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins

PubMed Central

2008-01-01

Background Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. Results A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. Conclusion The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated. Overall Hrs exhibit a considerable lack of evolutionary success in metazoans. PMID:18764950

Lack of adaptation to human tetherin in HIV-1 Group O and P

PubMed Central

2011-01-01

Background HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. Results We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. Conclusions The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread. PMID:21955466

[Improvement and the mechanism of cardiac function by knockdown of ADAM10 in adriamycin-induced cardiomyopathy rats].

PubMed

Li, Xiaoou; Xie, Lili; He, Bing; Huang, Wei

2018-01-01

Objective To study the role of a disintegrin and metalloproteinase10 (ADAM10) in shedding neural cadherin (N-cadherin) and develop an approach to interfere the process of ventricular remodeling in adriamycin-induced cardiomyopathy (ACM) rats. Methods In a rat model of ACM, the effects of intraperitoneal injection of the lentiviral RNAi vector of ADAM10 on the morphology of cardiomyocytes and contractile function were observed by HE staining and color Doppler echocardiography. The expressions of N-cadherin and C-terminal fragment 1 (CTF1) were detected by Western blotting and immunohistochemistry. Results In the in vivo experiment, a large amount of fluorescence was seen in the isolated primary cardiomyocytes, which indicated that the transfection in the rat model was successful. In the treatment group, the morphology of cardiomyocytes and function of the heart were evidently improved, N-cadherin protein expression was remarkably up-regulated and CTF1 protein was obviously down-regulated compared with the model group. Conclusion Knock-down of ADAM10 increases N-cadherin expression and decreases CTF1 expression, thus improves cardiac function in the rat model of ACM.

Role of a new Rho family member in cell migration and axon guidance in C. elegans.

PubMed

Zipkin, I D; Kindt, R M; Kenyon, C J

1997-09-05

Rho family GTPases are thought to regulate actin-dependent processes, but their functions in vivo are still poorly understood. We have investigated the function of a new, widely expressed Rho family member in C. elegans by analyzing mutations in the endogenous gene. Activated and null alleles all inhibit cell migration, demonstrating that this protein is required for cell migration in vivo. Only a small subset of the migrations inhibited by activating mutations are inhibited by null mutations, suggesting that considerable functional redundancy exists within this system. Our findings support this conclusion and show that mig-2 functions redundantly with another pathway to regulate nuclear migration. Surprisingly, activated alleles also cause misguided axon growth, suggesting that Rho family GTPases may couple guidance cues to process outgrowth.

Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly

PubMed Central

2014-01-01

Background Royal jelly (RJ) is a proteinaceous secretion produced from the hypopharyngeal and mandibular glands of nurse bees. It plays vital roles in honeybee biology and in the improvement of human health. However, some proteins remain unknown in RJ, and mapping N-glycosylation modification sites on RJ proteins demands further investigation. We used two different liquid chromatography-tandem mass spectrometry techniques, complementary N-glycopeptide enrichment strategies, and bioinformatic approaches to gain a better understanding of novel and glycosylated proteins in RJ. Results A total of 25 N-glycosylated proteins, carrying 53 N-glycosylation sites, were identified in RJ proteins, of which 42 N-linked glycosylation sites were mapped as novel on RJ proteins. Most of the glycosylated proteins were related to metabolic activities and health improvement. The 13 newly identified proteins were also mainly associated with metabolic processes and health improvement activities. Conclusion Our in-depth, large-scale mapping of novel glycosylation sites represents a crucial step toward systematically revealing the functionality of N-glycosylated RJ proteins, and is potentially useful for producing a protein with desirable pharmacokinetic and biological activity using a genetic engineering approach. The newly-identified proteins significantly extend the proteome coverage of RJ. These findings contribute vital and new knowledge to our understanding of the innate biochemical nature of RJ at both the proteome and glycoproteome levels. PMID:24529077

Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

PubMed Central

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-01-01

Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

Differential proteome and gene expression for testis of mice exposed to carbon ion radiation

NASA Astrophysics Data System (ADS)

Zhang, Hong; Li, Hongyan

Objective To investigate the effect and mechanism of high linear energy transfer (LET) carbon ion irradiation (CIR) on reproduction in the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Male mice underwent whole-body irradiation with CIR (0.5, 1 and 4Gy), and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis was used to determine the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 7, 14 days. Results 15 differentially expressed proteins, such as glucose-regulated protein(GRP78), aconitate hydratase-mitochondrial precursor (ACO), pyruvate kinase isozymes M1/M2 (PKM1/M2), glutathione-S-transferaseA3 (GSTA3), glutathione S-transferase Pi 1 (GSTP1), Cu/Zn super-oxide dismutase (SOD1), Peptidyl-prolyl cis-trans isomerase (Pin1) and Heat shock 70 kDa protein 4L (HSPa4L), were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Conclusion The findings of the present study demonstrated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. These data also may provide a scientific basis for protecting astronauts and space traveler’s health and safety.

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

PubMed Central

Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

2015-01-01

Background: There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. Aims: To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Methods: Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. Results: We found a 20% decrease in NR1 protein (t(66)=−2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=−2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Conclusions: Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient. PMID:27336043

Structural and functional development of small intestine in intrauterine growth retarded porcine offspring born to gilts fed diets with differing protein ratios throughout pregnancy.

PubMed

Mickiewicz, M; Zabielski, R; Grenier, B; Le Normand, L; Savary, G; Holst, J J; Oswald, I P; Metges, C C; Guilloteau, P

2012-06-01

Protein level in the maternal diet plays a crucial role in fetal programming during pregnancy. Low or high protein level increases the risk of intrauterine growth retardation (IUGR). The aim of this study was to investigate the structural and functional development of the small intestine in piglets from sows fed a control (C, 12.1% protein), a high protein (HP, 30% protein), or a low protein (LP, 6.5% protein) diet during pregnancy. Newborns were classified as IUGR (birth weight ≤1.18 kg) and non-IUGR (birth weight >1.18 kg). The piglets were euthanized on postnatal day (PD)1, PD28 and PD188. The LP diet in non-IUGR neonates resulted in decreased body weight on PD1. The LP and HP diets resulted in both decreased body weight and delayed catch-up growth in the IUGR piglets. The HP and LP-diets increased the length of villi on PD1 in non-IUGRs but not in IUGRs. At birth, the expressions of Ki67 and active caspase 3 in mid-jejunum epithelium of HP and LP non-IUGR neonates were significantly lower as compared to C non-IUGRs whilst in IUGRs the respective expressions were as high as in C non-IUGRs. The postnatal dynamics of brush border enzyme activities and vacuolated enterocytes disappearance showed significant drop in enterocyte maturation in IUGR as compared to non-IUGR neonates. In conclusion, both HP and LP diets led to retarded development of non-IUGR piglets. In IUGR piglets both HP and LP diets resulted in delayed catch-up growth, without adaptive changes in brush border digestive enzymes.

A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration

PubMed Central

Zhang, Huiliang; Wang, Pei; Bisetto, Sara; Yoon, Yisang; Chen, Quan; Sheu, Shey-Shing; Wang, Wang

2017-01-01

Aims Mitochondria in adult cardiomyocytes exhibit static morphology and infrequent dynamic changes, despite the high abundance of fission and fusion regulatory proteins in the heart. Previous reports have indicated that fusion proteins may bear functions beyond morphology regulation. Here, we investigated the role of fission protein, dynamin-related protein 1 (DRP1), on mitochondrial respiration regulation in adult cardiomyocytes. Methods and results By using genetic or pharmacological approaches, we manipulated the activity or protein level of fission and fusion proteins and found they mildly influenced mitochondrial morphology in adult rodent cardiomyocytes, which is in contrast to their significant effect in H9C2 cardiac myoblasts. Intriguingly, inhibiting endogenous DRP1 by dominant-negative DRP1 mutation (K38A), shRNA, or Mdivi-1 suppressed maximal respiration and respiratory control ratio in isolated mitochondria from adult mouse heart or in adult cardiomyocytes from rat. Meanwhile, basal respiration was increased due to increased proton leak. Facilitating mitofusin-mediated fusion by S3 compound, however, failed to inhibit mitochondrial respiration in adult cardiomyocytes. Mechanistically, DRP1 inhibition did not affect the maximal activity of individual respiratory chain complexes or the assembly of supercomplexes. Knocking out cyclophilin D, a regulator of mitochondrial permeability transition pore (mPTP), abolished the effect of DRP1 inhibition on respiration. Finally, DRP1 inhibition decreased transient mPTP-mediated mitochondrial flashes, delayed laser-induced mPTP opening and suppressed mitochondrial reactive oxygen species (ROS). Conclusion These results uncover a novel non-canonical function of the fission protein, DRP1 in maintaining or positively stimulating mitochondrial respiration, bioenergetics and ROS signalling in adult cardiomyocyte, which is likely independent of morphological changes. PMID:27794519

Commensurate distances and similar motifs in genetic congruence and protein interaction networks in yeast

PubMed Central

Ye, Ping; Peyser, Brian D; Spencer, Forrest A; Bader, Joel S

2005-01-01

Background In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways. Results We have defined congruent genes as pairs of genes with similar sets of genetic interaction partners and constructed a genetic congruence network by linking congruent genes. By comparing path lengths in three types of networks (genetic interaction, genetic congruence, and protein interaction), we discovered that high genetic congruence not only exhibits correlation with direct protein interaction linkage but also exhibits commensurate distance with the protein interaction network. However, consistent distances were not observed between genetic and protein interaction networks. We also demonstrated that congruence and protein networks are enriched with motifs that indicate network transitivity, while the genetic network has both transitive (triangle) and intransitive (square) types of motifs. These results suggest that robustness of yeast cells to gene deletions is due in part to two complementary pathways (square motif) or three complementary pathways, any two of which are required for viability (triangle motif). Conclusion Genetic congruence is superior to genetic interaction in prediction of protein interactions and function associations. Genetically interacting pairs usually belong to parallel compensatory pathways, which can generate transitive motifs (any two of three pathways needed) or intransitive motifs (either of two pathways needed). PMID:16283923

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

PubMed Central

2014-01-01

Background The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome. PMID:24987490

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell

PubMed Central

2010-01-01

Background The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. Results Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains. Conclusion This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions. PMID:21050442

Septins - active GTPases or just GTP-binding proteins?

PubMed

Abbey, Megha; Gaestel, Matthias; Menon, Manoj B

2018-05-10

Septins are conserved cytoskeletal proteins with unique filament forming capabilities and roles in cytokinesis and cell morphogenesis. Septins undergo hetero-oligomerization and assemble into higher order structures including filaments, rings and cages. Hetero- and homotypic interactions of septin isoforms involve alternating GTPase (G)-domain interfaces and those mediated by N- and C-terminal extensions. While most septins bind GTP, display weak GTP-hydrolysis activity and incorporate guanine nucleotides in their interaction interfaces, studies using GTPase-inactivating mutations have failed to conclusively establish a crucial role for GTPase activity in mediating septin functions. In this mini-review, we will critically assess the role of GTP-binding and -hydrolysis on septin assembly and function. The relevance of G-domain activity will also be discussed in the context of human septin mutations as well as the development of specific small-molecules targeting septin polymerization. As structural determinants of septin oligomer interfaces, G-domains are attractive targets for ligand-based inhibition of septin assembly. Whether such an intervention can predictably alter septin function is a major question for future research. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Mining for class-specific motifs in protein sequence classification

PubMed Central

2013-01-01

Background In protein sequence classification, identification of the sequence motifs or n-grams that can precisely discriminate between classes is a more interesting scientific question than the classification itself. A number of classification methods aim at accurate classification but fail to explain which sequence features indeed contribute to the accuracy. We hypothesize that sequences in lower denominations (n-grams) can be used to explore the sequence landscape and to identify class-specific motifs that discriminate between classes during classification. Discriminative n-grams are short peptide sequences that are highly frequent in one class but are either minimally present or absent in other classes. In this study, we present a new substitution-based scoring function for identifying discriminative n-grams that are highly specific to a class. Results We present a scoring function based on discriminative n-grams that can effectively discriminate between classes. The scoring function, initially, harvests the entire set of 4- to 8-grams from the protein sequences of different classes in the dataset. Similar n-grams of the same size are combined to form new n-grams, where the similarity is defined by positive amino acid substitution scores in the BLOSUM62 matrix. Substitution has resulted in a large increase in the number of discriminatory n-grams harvested. Due to the unbalanced nature of the dataset, the frequencies of the n-grams are normalized using a dampening factor, which gives more weightage to the n-grams that appear in fewer classes and vice-versa. After the n-grams are normalized, the scoring function identifies discriminative 4- to 8-grams for each class that are frequent enough to be above a selection threshold. By mapping these discriminative n-grams back to the protein sequences, we obtained contiguous n-grams that represent short class-specific motifs in protein sequences. Our method fared well compared to an existing motif finding method known as Wordspy. We have validated our enriched set of class-specific motifs against the functionally important motifs obtained from the NLSdb, Prosite and ELM databases. We demonstrate that this method is very generic; thus can be widely applied to detect class-specific motifs in many protein sequence classification tasks. Conclusion The proposed scoring function and methodology is able to identify class-specific motifs using discriminative n-grams derived from the protein sequences. The implementation of amino acid substitution scores for similarity detection, and the dampening factor to normalize the unbalanced datasets have significant effect on the performance of the scoring function. Our multipronged validation tests demonstrate that this method can detect class-specific motifs from a wide variety of protein sequence classes with a potential application to detecting proteome-specific motifs of different organisms. PMID:23496846

The application of multiple reaction monitoring and multi-analyte profiling to HDL proteins

PubMed Central

2014-01-01

Background HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD). Methods We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls. Results We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP. Conclusions MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies. PMID:24397693

Discovery of a Distinct Superfamily of Kunitz-Type Toxin (KTT) from Tarantulas

PubMed Central

Diao, Jian-Bo; Jiang, Li-Ping; Tang, Xing; Liang, Song-Ping

2008-01-01

Background Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking) were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle. Principal Findings Here we report the presence of a new superfamily of KTTs in spiders (Tarantulas: Ornithoctonus huwena and Ornithoctonus hainana), which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI) as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (ω) for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins) derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications. Conclusions/Significance These finding illustrate that the two activity sites of Kunitz-type toxins are functionally and evolutionally independent and provide new insights into effects of Darwinian selection pressures on KTT evolution, and mechanisms by which new functions can be grafted onto old protein scaffolds. PMID:18923708

Expression of the Hsp23 chaperone during Drosophila embryogenesis: association to distinct neural and glial lineages

PubMed Central

Michaud, Sébastien; Tanguay, Robert M

2003-01-01

Background In addition to their strong induction following stress, small heat shock proteins (Hsp) are also expressed during development in a wide variety of organisms. However, the precise identity of cell(s) expressing these proteins and the functional contribution of small heat shock proteins in such developmental context remain to be determined. The present study provides a detailed description of the Drosophila small heat shock protein Hsp23 expression pattern during embryogenesis and evaluates its functional contribution to central nervous system development. Results Throughout embryogenesis, Hsp23 is expressed in a stage-specific manner by a restricted number of neuronal and glial lineages of the central nervous system. Hsp23 is also detected in the amnioserosa and within a single lateral chordotonal organ. Its expression within the MP2 lineage does not require the presence of a functional midline nor the activity of the Notch signaling pathway. Transactivation assays demonstrate that transcription factors implicated in the differentiation of the midline also regulate hsp23 promoter activity. Phenotypic analysis of a transgenic line exhibiting loss of Hsp23 expression in the central nervous system suggests that Hsp23 is not required for development and function of this tissue. Likewise, its overexpression does not cause deleterious effects, as development remains unaffected. Conclusions Based on the presented data, we suggest that the tightly regulated developmental expression of Hsp23 is not actively involved in cell differentiation and central nervous system development per se but rather reflects a putative role in preventive "pre-stress" neuroprotection or in non-vital process(es) common to the identified cell lineages. PMID:14617383

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels

PubMed Central

Liu, Wei; Hu, Dong; Huo, Haizhong; Zhang, Weifeng; Adiliaghdam, Fatemeh; Morrison, Sarah; Ramirez, Juan M; Gul, Sarah S; Hamarneh, Sulaiman R; Hodin, Richard A

2017-01-01

BACKGROUND Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. STUDY DESIGN The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). RESULTS Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP over-expression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. CONCLUSIONS Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function. PMID:27106638

New Model of Action for Mood Stabilizers: Phosphoproteome from Rat Pre-Frontal Cortex Synaptoneurosomal Preparations

PubMed Central

Corena-McLeod, Maria; Walss-Bass, Consuelo; Oliveros, Alfredo; Gordillo Villegas, Andres; Ceballos, Carolina; Charlesworth, Cristine M.; Madden, Benjamin; Linser, Paul J.; Van Ekeris, Leslie; Smith, Kristin; Richelson, Elliott

2013-01-01

Background Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level. Hypothesis Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement. Methods Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry. Results Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation. Conclusions Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs. PMID:23690912

The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

PubMed Central

Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

2013-01-01

BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

Lithostathine, the presumed pancreatic stone inhibitor, does not interact specifically with calcium carbonate crystals.

PubMed

De Reggi, M; Gharib, B; Patard, L; Stoven, V

1998-02-27

Lithostathine (pancreatic stone protein, Reg protein) is, in addition to albumin, the major nonenzymatic protein of the pancreatic juice. It has been assumed to inhibit calcium carbonate precipitation and therefore to prevent stone formation in the pancreatic ducts. This function is, however, debatable. The assumption is based on the inhibition of in vitro crystal nucleation and growth by lithostathine. Considering that these phenomena occur only under certain critical conditions, we re-examined the question using a protein preparation where the purity and folding have been tested by mass spectroscopy and NMR in the absence of nonprotein contaminants. Under these conditions, we showed conclusively that lithostathine does not inhibit calcium carbonate nucleation and crystal growth. We demonstrated that previous findings on the alleged inhibition can be attributed to the uncontrolled presence of salts in the protein preparation used. Moreover, the affinity of lithostathine to calcite crystals, expressed as the half-life of bound iodinated protein in the presence of unlabeled competitor, was significantly lower than that of bovine serum albumin (8.8 and 11.2 h, respectively). Using glass microspheres instead of crystals did not significantly change the half-life of bound lithostathine (8.0 h). These findings are incompatible with the hypothesis of a specific interaction of lithostathine with calcium carbonate crystals. In conclusion, considering that components of pancreatic juice such as NaCl and phosphate ions are powerful inhibitors of calcium carbonate crystal growth, the mechanism of stone formation in pancreatic ducts must be reconsidered. The presence in normal pancreatic juice of small amounts of the 133-residue isoform of lithostathine (PSP-S1), which precipitates at physiological pH, should be noted, and the possibility should be considered that they form micro-precipitates that aggregate and are progressively calcified.

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

PubMed Central

Wulfkuhle, Julia D.; Berg, Daniela; Wolff, Claudia; Langer, Rupert; Tran, Kai; Illi, Julie; Espina, Virginia; Pierobon, Mariaelena; Deng, Jianghong; DeMichele, Angela; Walch, Axel; Bronger, Holger; Becker, Ingrid; Waldhör, Christine; Höfler, Heinz; Esserman, Laura; Liotta, Lance A.; Becker, Karl-Friedrich; Petricoin, Emanuel F.

2017-01-01

Purpose Targeting of the HER2 protein in human breast cancer represents a major advance in oncology, but relies on measurements of total HER2 protein and not HER2 signaling network activation. We utilized reverse phase protein microarrays (RPMAs) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Experimental Design Three independent study sets, comprising a total of 415 individual patient samples from flash frozen core biopsy samples and FFPE surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pre-treatment core biopsy samples and whole tissue lysates from 288 FFPE samples and these results were compared to FISH and IHC. Results RPMA measurements of total HER2 were highly concordant (> 90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC+ population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC- tumors and, identical to that seen with FISH/IHC+ tumors, the HER2 activation was concordant with EGFR and HER3 phosphorylation and downstream signaling endpoint activation. Conclusions Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC-/FISH-/pHER2+ tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR. PMID:23045247

Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors

PubMed Central

Zientara-Rytter, Katarzyna; Łukomska, Jolanta; Moniuszko, Grzegorz; Gwozdecki, Rafał; Surowiecki, Przemysław; Lewandowska, Małgorzata; Liszewska, Frantz; Wawrzyńska, Anna

2011-01-01

Two main mechanisms of protein turnover exist in eukaryotic cells: the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Autophagy is an emerging important constituent of many physiological and pathological processes, such as response to nutrient deficiency, programmed cell death and innate immune response. In mammalian cells the selectivity of autophagy is ensured by the presence of cargo receptors, such as p62/SQSTM1 and NBR1, responsible for sequestration of the ubiquitinated proteins. In plants no selective cargo receptors have been identified yet. The present report indicates that structural and functional homologs of p62 and NBR1 proteins exist in plants. The tobacco protein, named Joka2, has been identified in yeast two-hybrid search as a binding partner of a small coiled-coil protein, a member of UP9/LSU family of unknown function, encoded by the UP9C gene strongly and specifically induced during sulfur deficiency. The typical domains of p62 and NBR1 are conserved in Joka2. Similarly to p62, Joka2-YFP has dual localization (cytosolic speckles and the nucleus); it forms homodimers and interacts with a member of the ATG8 family. Increased expression of Joka2 and ATG8f was observed in roots of tobacco plants grown for two days in nutrient-deficient conditions. Constitutive ectopic expression of Joka2-YFP in tobacco resulted in attenuated response (manifested by lesser yellowing of the leaves) to nutrient deficiency. In conclusion, Joka2, and presumably the process of selective autophagy, might constitute an important part of plant response to environmental stresses. PMID:21670587

CSF N-glycan profile reveals sialylation deficiency in a patient with GM2 gangliosidosis presenting as childhood disintegrative disorder.

PubMed

Barone, Rita; Sturiale, Luisella; Fiumara, Agata; Palmigiano, Angelo; Bua, Rosaria O; Rizzo, Renata; Zappia, Mario; Garozzo, Domenico

2016-04-01

Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2-gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

BKV Agnoprotein Interacts with α-Soluble N-Ethylmaleimide-Sensitive Fusion Attachment Protein, and Negatively Influences Transport of VSVG-EGFP

PubMed Central

Johannessen, Mona; Walquist, Mari; Gerits, Nancy; Dragset, Marte; Spang, Anne; Moens, Ugo

2011-01-01

Background The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. Principal Findings As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them α-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and α-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and α-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with α-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. Conclusions We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with α-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter. PMID:21931730

R516Q mutation in Melanoma differentiation-associated protein 5 (MDA5) and its pathogenic role towards rare Singleton-Merten syndrome; a signature associated molecular dynamics study.

PubMed

Raghuraman, P; Sudandiradoss, C

2018-02-20

Singleton-Merten syndrome, a critical and rare multifactorial disorder that is closely linked to R516Q mutation in MDA5 protein associated with an enhanced interferon response in the affected individual. In the present study, we provide conclusive key evidence on R516Q mutation and their connectivity towards sequence-structural basis dysfunction of MDA5 protein. Among the various mutations, we found R516Q is the most pathogenic mutation based on mutational signature Q-A-[RE]-G-R-[GA]-R-A-[ED]-[DE]-S-[ST]-Y-[TSAV]-L-V designed from our work. Further, we derived a distant ortholog for this mutational signature from which we identified 343 intra-residue interactions that fall communally in the position required to maintain the structural and functional integration of protein architecture. This identification served us to understand the critical role of hot spots in residual aggregation that holds a native form of folding conformation in the functional region. In addition, the long-range molecular dynamics simulation demarcated the residual dependencies of conformational transition in distinct regions ( L29 360-370 α18 , α19 380-410 L31 , α21 430-480 L33-α22-L35 and α24 510-520 L38 ) occurring upon R516Q mutation. Together, our results emphasise that the dislocation of functional hot spots Pro229, Arg414, Val498, Met510, Ala513, Gly515 and Arg516 in MDA5 protein which is important for interior structural packing and fold arrangements. In a nutshell, our findings are perfectly conceded with other experimental reports and will have potential implications in immune therapeutical advancement for rare singleton-merten syndrome.

The High Mobility Group A1 (HMGA1) Transcriptome in Cancer and Development

PubMed Central

Sumter, T.F.; Xian, L.; Huso, T.; Koo, M.; Chang, Y.-T.; Almasri, T.N.; Chia, L.; Inglis, C.; Reid, D.; Resar, L.M.S.

2017-01-01

Background & Objectives Chromatin structure is the single most important feature that distinguishes a cancer cell from a normal cell histologically. Chromatin remodeling proteins regulate chromatin structure and high mobility group A (HMGA1) proteins are among the most abundant, nonhistone chromatin remodeling proteins found in cancer cells. These proteins include HMGA1a/HMGA1b isoforms, which result from alternatively spliced mRNA. The HMGA1 gene is overexpressed in cancer and high levels portend a poor prognosis in diverse tumors. HMGA1 is also highly expressed during embryogenesis and postnatally in adult stem cells. Overexpression of HMGA1 drives neoplastic transformation in cultured cells, while inhibiting HMGA1 blocks oncogenic and cancer stem cell properties. Hmga1 transgenic mice succumb to aggressive tumors, demonstrating that dysregulated expression of HMGA1 causes cancer in vivo. HMGA1 is also required for reprogramming somatic cells into induced pluripotent stem cells. HMGA1 proteins function as ancillary transcription factors that bend chromatin and recruit other transcription factors to DNA. They induce oncogenic transformation by activating or repressing specific genes involved in this process and an HMGA1 “transcriptome” is emerging. Although prior studies reveal potent oncogenic properties of HMGA1, we are only beginning to understand the molecular mechanisms through which HMGA1 functions. In this review, we summarize the list of putative downstream transcriptional targets regulated by HMGA1. We also briefly discuss studies linking HMGA1 to Alzheimer’s disease and type-2 diabetes. Conclusion Further elucidation of HMGA1 function should lead to novel therapeutic strategies for cancer and possibly for other diseases associated with aberrant HMGA1 expression. PMID:26980699

Recognition deficits in mice carrying mutations of genes encoding BLOC-1 subunits pallidin or dysbindin.

PubMed

Spiegel, S; Chiu, A; James, A S; Jentsch, J D; Karlsgodt, K H

2015-11-01

Numerous studies have implicated DTNBP1, the gene encoding dystrobrevin-binding protein or dysbindin, as a candidate risk gene for schizophrenia, though this relationship remains somewhat controversial. Variation in dysbindin, and its location on chromosome 6p, has been associated with cognitive processes, including those relying on a complex system of glutamatergic and dopaminergic interactions. Dysbindin is one of the seven protein subunits that comprise the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Dysbindin protein levels are lower in mice with null mutations in pallidin, another gene in the BLOC-1, and pallidin levels are lower in mice with null mutations in the dysbindin gene, suggesting that multiple subunit proteins must be present to form a functional oligomeric complex. Furthermore, pallidin and dysbindin have similar distribution patterns in a mouse and human brain. Here, we investigated whether the apparent correspondence of pallid and dysbindin at the level of gene expression is also found at the level of behavior. Hypothesizing a mutation leading to underexpression of either of these proteins should show similar phenotypic effects, we studied recognition memory in both strains using the novel object recognition task (NORT) and social novelty recognition task (SNRT). We found that mice with a null mutation in either gene are impaired on SNRT and NORT when compared with wild-type controls. These results support the conclusion that deficits consistent with recognition memory impairment, a cognitive function that is impaired in schizophrenia, result from either pallidin or dysbindin mutations, possibly through degradation of BLOC-1 expression and/or function. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Expression of Pleiotrophin in the Prostate is Androgen Regulated and it Functions as an Autocrine Regulator of Mesenchyme and Cancer Associated Fibroblasts and as a Paracrine Regulator of Epithelia

PubMed Central

Orr, Brigid; Vanpoucke, Griet; Grace, O Cathal; Smith, Lee; Anderson, Richard A; Riddick, Antony CP; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

2011-01-01

BACKGROUND Androgens and paracrine signaling from mesenchyme/stroma regulate development and disease of the prostate, and gene profiling studies of inductive prostate mesenchyme have identified candidate molecules such as pleiotrophin (Ptn). METHODS Ptn transcripts and protein were localized by in situ and immunohistochemistry and Ptn mRNA was quantitated by Northern blot and qRT-PCR. Ptn function was examined by addition of hPTN protein to rat ventral prostate organ cultures, primary human fetal prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. RESULTS During development, Ptn transcripts and protein were expressed in ventral mesenchymal pad (VMP) and prostatic mesenchyme. Ptn was localized to mesenchyme surrounding ductal epithelial tips undergoing branching morphogenesis, and was located on the surface of epithelia. hPTN protein stimulated branching morphogenesis and stromal and epithelial proliferation, when added to rat VP cultures, and also stimulated growth of fetal human prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. PTN mRNA was enriched in patient-matched normal prostate fibroblasts versus prostate cancer associated fibroblasts. PTN also showed male enriched expression in fetal human male urethra versus female, and between wt male and ARKO male mice. Transcripts for PTN were upregulated by testosterone in fetal human prostate fibroblasts and organ cultures of female rat VMP. Ptn protein was increased by testosterone in organ cultures of female rat VMP and in rat male urethra compared to female. CONCLUSIONS Our data suggest that in the prostate Ptn functions as a regulator of both mesenchymal and epithelial proliferation, and that androgens regulate Ptn levels. Prostate 71:305–317, 2011. © 2010 Wiley-Liss, Inc. PMID:20812209

Functional Relevance of Protein Glycosylation to the Pro-Inflammatory Effects of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) on Monocytes/Macrophages

PubMed Central

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Background and Objective Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. Methods The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. Results 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Conclusions Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages. PMID:25658763

Chronic exposure to emissions from photocopiers in copy shops causes oxidative stress and systematic inflammation among photocopier operators in India

PubMed Central

2013-01-01

Background We assessed indoor air quality in photocopier centers and investigated whether occupational exposure to emissions from photocopiers is associated with decline in lung function or changes in haematological parameters, oxidative stress and inflammatory status. Methods Indoor air quality was monitored in five photocopier centers. Pulmonary function was assessed by spirometry in 81 photocopier operators (64 male and 17 female) and 43 healthy control (31 male and 12 female) subjects. Hematological status, serum thio-barbituric acid reactive substances (TBARS), total ferric reducing antioxidant capacity (FRAC), leukotriene B4 (LTB4), 8-isoprostane, C reactive protein (CRP), interleukin 8 (IL-8), clara cell protein (CC-16), intercellular adhesion molecule 1 (ICAM-1) and eosinophilic cationic protein (ECP) were analyzed. Relationships between cumulative exposure, lung function and inflammatory markers were assessed. Results PM10 and PM2.5 were above the permissible levels in all the photocopier centers, whereas the levels of carbon monoxide, nitrogen oxides, ozone, sulphur dioxide, lead, arsenic, nickel, ammonia, benzene and benzo(a)pyrene were within Indian ambient air quality standards. Lung function was similar in the photocopier operators and control subjects. Serum TBARS was significantly higher and FRAC was lower among photocopier operators when compared to healthy controls. Plasma IL-8, LTB4, ICAM-1 and ECP were significantly higher in the photocopier exposed group. Conclusions Photocopiers emit high levels of particulate matter. Long term exposure to emissions from photocopiers was not associated with decreased lung function, but resulted in high oxidative stress and systemic inflammation leading to high risk of cardiovascular diseases. PMID:24025094

Protein–Protein Interactions in Dilute to Concentrated Solutions: α-Chymotrypsinogen in Acidic Conditions

PubMed Central

2015-01-01

Protein–protein interactions were investigated for α-chymotrypsinogen by static and dynamic light scattering (SLS and DLS, respectively), as well as small-angle neutron scattering (SANS), as a function of protein and salt concentration at acidic conditions. Net protein–protein interactions were probed via the Kirkwood–Buff integral G22 and the static structure factor S(q) from SLS and SANS data. G22 was obtained by regressing the Rayleigh ratio versus protein concentration with a local Taylor series approach, which does not require one to assume the underlying form or nature of intermolecular interactions. In addition, G22 and S(q) were further analyzed by traditional methods involving fits to effective interaction potentials. Although the fitted model parameters were not always physically realistic, the numerical values for G22 and S(q → 0) were in good agreement from SLS and SANS as a function of protein concentration. In the dilute regime, fitted G22 values agreed with those obtained via the osmotic second virial coefficient B22 and showed that electrostatic interactions are the dominant contribution for colloidal interactions in α-chymotrypsinogen solutions. However, as protein concentration increases, the strength of protein–protein interactions decreases, with a more pronounced decrease at low salt concentrations. The results are consistent with an effective “crowding” or excluded volume contribution to G22 due to the long-ranged electrostatic repulsions that are prominent even at the moderate range of protein concentrations used here (<40 g/L). These apparent crowding effects were confirmed and quantified by assessing the hydrodynamic factor H(q → 0), which is obtained by combining measurements of the collective diffusion coefficient from DLS data with measurements of S(q → 0). H(q → 0) was significantly less than that for a corresponding hard-sphere system and showed that hydrodynamic nonidealities can lead to qualitatively incorrect conclusions regarding B22, G22, and static protein–protein interactions if one uses only DLS to assess protein interactions. PMID:24810917

In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea

PubMed Central

2012-01-01

Background Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell. Results Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes. Conclusions The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs. PMID:22540284

Triangle network motifs predict complexes by complementing high-error interactomes with structural information

PubMed Central

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-01-01

Background A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. Results We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Conclusion Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN. PMID:19558694

Proteome analysis of plastids from developing seeds of Jatropha curcas L.

PubMed

Pinheiro, Camila B; Shah, Mohibullah; Soares, Emanoella L; Nogueira, Fábio C S; Carvalho, Paulo C; Junqueira, Magno; Araújo, Gabriel D T; Soares, Arlete A; Domont, Gilberto B; Campos, Francisco A P

2013-11-01

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

PubMed

Wang, Yaxi; Sun, Tingyi; Sun, Haimei; Yang, Shu; Li, Dandan; Zhou, Deshan

2017-04-06

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Screening and Evaluation of Deleterious SNPs in APOE Gene of Alzheimer's Disease.

PubMed

Masoodi, Tariq Ahmad; Al Shammari, Sulaiman A; Al-Muammar, May N; Alhamdan, Adel A

2012-01-01

Introduction. Apolipoprotein E (APOE) is an important risk factor for Alzheimer's disease (AD) and is present in 30-50% of patients who develop late-onset AD. Several single-nucleotide polymorphisms (SNPs) are present in APOE gene which act as the biomarkers for exploring the genetic basis of this disease. The objective of this study is to identify deleterious nsSNPs associated with APOE gene. Methods. The SNPs were retrieved from dbSNP. Using I-Mutant, protein stability change was calculated. The potentially functional nonsynonymous (ns) SNPs and their effect on protein was predicted by PolyPhen and SIFT, respectively. FASTSNP was used for functional analysis and estimation of risk score. The functional impact on the APOE protein was evaluated by using Swiss PDB viewer and NOMAD-Ref server. Results. Six nsSNPs were found to be least stable by I-Mutant 2.0 with DDG value of >-1.0. Four nsSNPs showed a highly deleterious tolerance index score of 0.00. Nine nsSNPs were found to be probably damaging with position-specific independent counts (PSICs) score of ≥2.0. Seven nsSNPs were found to be highly polymorphic with a risk score of 3-4. The total energies and root-mean-square deviation (RMSD) values were higher for three mutant-type structures compared to the native modeled structure. Conclusion. We concluded that three nsSNPs, namely, rs11542041, rs11542040, and rs11542034, to be potentially functional polymorphic.

Stability of lutein encapsulated whey protein nano-emulsion during storage

PubMed Central

Guo, Mingruo

2018-01-01

Lutein is a hydrophobic carotenoid that has multiple health functions. However, the application of lutein is limited due to its poor solubility in water and instability under certain conditions during storage. Hereby we generated lutein loaded nano-emulsions using whey protein isolate (WPI) or polymerized whey protein isolate (PWP) with assistance of high intensity ultrasound and evaluate their stability during storage at different conditions. We measured the particle size, zeta-potential, physical stability and lutein content change. Results showed that the PWP based nano-emulsion system was not stable in the tested Oil/Water/Ethanol system indicated by the appearance of stratification within only one week. The WPI based nano-emulsion system showed stable physiochemical stability during the storage at 4°C. The lutein content of the system was reduced by only 4% after four weeks storage at 4°C. In conclusion, our whey protein based nano-emulsion system provides a promising strategy for encapsulation of lutein or other hydrophobic bioactive molecules to expand their applications. PMID:29415071

The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

PubMed

Mauney, Christopher H; Rogers, LeAnn C; Harris, Reuben S; Daniel, Larry W; Devarie-Baez, Nelmi O; Wu, Hanzhi; Furdui, Cristina M; Poole, Leslie B; Perrino, Fred W; Hollis, Thomas

2017-12-01

Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.

Biological role and structural mechanism of twinfilin–capping protein interaction

PubMed Central

Falck, Sandra; Paavilainen, Ville O; Wear, Martin A; Grossmann, J Günter; Cooper, John A; Lappalainen, Pekka

2004-01-01

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin–CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics. PMID:15282541

Structure-Function Analysis of Rgs1 in Magnaporthe oryzae: Role of DEP Domains in Subcellular Targeting

PubMed Central

Ramanujam, Ravikrishna; Yishi, Xu; Liu, Hao; Naqvi, Naweed I.

2012-01-01

Background Rgs1, a prototypical Regulator of G protein Signaling, negatively modulates the cyclic AMP pathway thereby influencing various aspects of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. Rgs1 possesses tandem DEP motifs (termed DEP-A and DEP-B; for Dishevelled, Egl-10, Pleckstrin) at the N-terminus, and a Gα-GTP interacting RGS catalytic core domain at the C-terminus. In this study, we focused on gaining further insights into the mechanisms of Rgs1 regulation and subcellular localization by characterizing the role(s) of the individual domains and the full-length protein during asexual development and pathogenesis in Magnaporthe. Methodology/Principal Findings Utilizing western blot analysis and specific antisera against the N- and C-terminal halves of Rgs1, we identify and report the in vivo endoproteolytic processing/cleavage of full-length Rgs1 that yields an N-terminal DEP and a RGS core domain. Independent expression of the resultant DEP-DEP half (N-Rgs1) or RGS core (C-Rgs1) fragments, failed to complement the rgs1Δ defects in colony morphology, aerial hyphal growth, surface hydrophobicity, conidiation, appressorium formation and infection. Interestingly, the full-length Rgs1-mCherry, as well as the tagged N-terminal DEP domains (individually or in conjunction) localized to distinct punctate vesicular structures in the cytosol, while the catalytic RGS core motif was predominantly vacuolar. Conclusions/Significance Based on our data from sequence alignments, immuno-blot and microscopic analysis, we propose that the post-translational proteolytic processing of Rgs1 and the vacuolar sequestration of the catalytic RGS domain represents an important means of down regulating Rgs1 function and thus forming an additional and alternative means of regulating G protein signaling in Magnaporthe. We further hypothesize the prevalence of analogous mechanisms functioning in other filamentous fungi. Furthermore, we conclusively assign a specific vesicular/membrane targeting function for the N-terminal DEP domains of Rgs1 in the rice-blast fungus. PMID:22927898

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

PubMed Central

2014-01-01

Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

A facile approach to the isolation of proteins in Ferula asafoetida and their enzyme stabilizing, anti-microbial and anti-oxidant activity.

PubMed

Chandran, Sanjana; Sakthivel, Meenakumari; Thirumavalavan, Munusamy; Thota, Jagadeshwar Reddy; Mariappanadar, Vairamani; Raman, Pachaiappan

2017-09-01

The objective of the present study was to identify the proteome pattern, isolate and study the functions of selective proteins from Ferula asafoetida root exudate using chromatographic techniques. The root exudate proteins were fractionated using ion-exchange and gel filtration chromatography. A range of bioactive protein fractions were then separated in sufficient quantity which is the focus of this study. Based on studies, here we report three main proteins with molecular weights 14kDa, 27kDa, and 39kDa. The biological and pharmacological activities of both purified and unpurified proteins obtained were extensively studied to understand their significance. The study revelaed that 27kDa protein interestingly stabilized trypsin activity in 24h of time and retained about 64% of the enzyme activity. Analyses confirmed 40°C and pH 8.0 are the optimum temperature and pH respectively. The 39kDa protein remarkably increased the activity of chymotrypsin and the 14kDa protein showed anti-bacterial activity against Pseudomonas aeruginosa. Invariably all of the three purified proteins showed enhanced anti-oxidant activity. In conclusion, results here obtained suggested that the primary metabolites (proteins) in asafoetida are mainly responsible for its versatile biological and pharmacological activities. Copyright © 2017 Elsevier B.V. All rights reserved.

The Spinal Antinociceptive Effects of Endomorphins in Rats: Behavioral and G Protein Functional Studies

PubMed Central

Xie, Hong; Woods, James H.; Traynor, John R.; Ko, Mei-Chuan

2008-01-01

BACKGROUND Endomorphin-1 and endomorphin-2 are endogenous peptides that are highly selective for μ-opioid receptors. However, studies of their functional efficacy and selectivity are controversial. In this study, we systematically compared the effects of intrathecal (i.t.) administration of endomorphin-1 and -2 on nociception assays and G protein activation with those of [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a highly effective peptidic μ-opioid receptor agonist. METHODS Male Sprague-Dawley rats were used. Acute and inflammatory pain models were used to compare the duration and magnitude of antinociception. Agonist-stimulated [35S]GTPγS binding was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. In addition, antagonists selective for each receptor type were used to verify the functional selectivity of endomorphins in the rat spinal cord. RESULTS After i.t. administration, endomorphin-1 and -2 produced less antinociceptive effects than DAMGO in the model of acute pain. Concentration–response curves for DAMGO-, endomorphin-1-, and endomorphin-2-stimulated [35S]GTPγS binding revealed that both endomorphin-1 and -2 produced less G protein activation (i.e., approximately 50%–60%) than DAMGO did in the membranes of spinal cord and thalamus. In addition, i.t. endomorphin-induced antinociception was blocked by μ-opioid receptor selective dose of naltrexone (P < 0.05), but not by δ- and κ-opioid receptor antagonists, naltrindole and nor-binaltorphimine (P > 0.05). CONCLUSIONS Endomorphins are partial agonists for G protein activation at spinal and thalamic μ-opioid receptors. Both in vivo and in vitro measurements together suggest that DAMGO is more effective than endomorphins. Spinal endomorphins’ antinociceptive efficacy may range between 53% and 84% depending on the intensity and modality of the nociceptive stimulus. PMID:18499626

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

PubMed

Nguyen, Vi N; Park, Annsea; Xu, Anting; Srouji, John R; Brenner, Steven E; Kirsch, Jack F

2016-12-01

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat /K m values >10,000 M -1  s -1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat /K m values exhibited against these canonical substrates are well under 10 5 M -1  s -1 . By contrast, several Nudix enzymes show much larger k cat /K m values (in the range of 10 5 to >10 7 M -1  s -1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

PubMed

Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

2012-02-01

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function. Copyright © 2012 Elsevier Inc. All rights reserved.

Mouse Nuclear Myosin I Knock-Out Shows Interchangeability and Redundancy of Myosin Isoforms in the Cell Nucleus

PubMed Central

Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

2013-01-01

Background Nuclear myosin I (NM1) is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes. PMID:23593477

Association of Urinary Biomarkers of Inflammation, Injury, and Fibrosis with Renal Function Decline: The ACCORD Trial

PubMed Central

Nadkarni, Girish N.; Rao, Veena; Ismail-Beigi, Faramarz; Fonseca, Vivian A.; Shah, Sudhir V.; Simonson, Michael S.; Cantley, Lloyd; Devarajan, Prasad; Parikh, Chirag R.

2016-01-01

Background and objectives Current measures for predicting renal functional decline in patients with type 2 diabetes with preserved renal function are unsatisfactory, and multiple markers assessing various biologic axes may improve prediction. We examined the association of four biomarker-to-creatinine ratio levels (monocyte chemotactic protein-1, IL-18, kidney injury molecule-1, and YKL-40) with renal outcome. Design, setting, participants, & measurements We used a nested case-control design in the Action to Control Cardiovascular Disease Trial by matching 190 participants with ≥40% sustained eGFR decline over the 5-year follow-up period to 190 participants with ≤10% eGFR decline in a 1:1 fashion on key characteristics (age within 5 years, sex, race, baseline albumin-to-creatinine ratio within 20 μg/mg, and baseline eGFR within 10 ml/min per 1.73 m2), with ≤10% decline. We used a Mesoscale Multiplex Platform and measured biomarkers in baseline and 24-month specimens, and we examined biomarker associations with outcome using conditional logistic regression. Results Baseline and 24-month levels of monocyte chemotactic protein-1-to-creatinine ratio levels were higher for cases versus controls. The highest quartile of baseline monocyte chemotactic protein-1-to-creatinine ratio had fivefold greater odds, and each log increment had 2.27-fold higher odds for outcome (odds ratio, 5.27; 95% confidence interval, 2.19 to 12.71 and odds ratio, 2.27; 95% confidence interval, 1.44 to 3.58, respectively). IL-18-to-creatinine ratio, kidney injury molecule-1-to-creatinine ratio, and YKL-40-to-creatinine ratio were not consistently associated with outcome. C statistic for traditional predictors of eGFR decline was 0.70, which improved significantly to 0.74 with monocyte chemotactic protein-1-to-creatinine ratio. Conclusions Urinary monocyte chemotactic protein-1-to-creatinine ratio concentrations were strongly associated with sustained renal decline in patients with type 2 diabetes with preserved renal function. PMID:27189318

Domain duplication, divergence, and loss events in vertebrate Msx paralogs reveal phylogenomically informed disease markers

PubMed Central

Finnerty, John R; Mazza, Maureen E; Jezewski, Peter A

2009-01-01

Background Msx originated early in animal evolution and is implicated in human genetic disorders. To reconstruct the functional evolution of Msx and inform the study of human mutations, we analyzed the phylogeny and synteny of 46 metazoan Msx proteins and tracked the duplication, diversification and loss of conserved motifs. Results Vertebrate Msx sequences sort into distinct Msx1, Msx2 and Msx3 clades. The sister-group relationship between MSX1 and MSX2 reflects their derivation from the 4p/5q chromosomal paralogon, a derivative of the original "MetaHox" cluster. We demonstrate physical linkage between Msx and other MetaHox genes (Hmx, NK1, Emx) in a cnidarian. Seven conserved domains, including two Groucho repression domains (N- and C-terminal), were present in the ancestral Msx. In cnidarians, the Groucho domains are highly similar. In vertebrate Msx1, the N-terminal Groucho domain is conserved, while the C-terminal domain diverged substantially, implying a novel function. In vertebrate Msx2 and Msx3, the C-terminal domain was lost. MSX1 mutations associated with ectodermal dysplasia or orofacial clefting disorders map to conserved domains in a non-random fashion. Conclusion Msx originated from a MetaHox ancestor that also gave rise to Tlx, Demox, NK, and possibly EHGbox, Hox and ParaHox genes. Duplication, divergence or loss of domains played a central role in the functional evolution of Msx. Duplicated domains allow pleiotropically expressed proteins to evolve new functions without disrupting existing interaction networks. Human missense sequence variants reside within evolutionarily conserved domains, likely disrupting protein function. This phylogenomic evaluation of candidate disease markers will inform clinical and functional studies. PMID:19154605

c-Myc Alters Substrate Utilization and O-GlcNAc Protein Posttranslational Modifications without Altering Cardiac Function during Early Aortic Constriction

PubMed Central

Ledee, Dolena; Smith, Lincoln; Bruce, Margaret; Kajimoto, Masaki; Isern, Nancy; Portman, Michael A.; Olson, Aaron K.

2015-01-01

Hypertrophic stimuli cause transcription of the proto-oncogene c-Myc (Myc). Prior work showed that myocardial knockout of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we assessed the interplay between Myc, substrate oxidation and cardiac function during early pressure overload hypertrophy. Mice with cardiac specific, inducible Myc knockout (MycKO-TAC) and non-transgenic littermates (Cont-TAC) were subjected to transverse aortic constriction (TAC; n = 7/group). Additional groups underwent sham surgery (Cont-Sham and MycKO-Sham, n = 5 per group). After two weeks, function was measured in isolated working hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. In sham hearts, Myc knockout did not affect cardiac function or substrate preferences for the citric acid cycle. However, Myc knockout altered fractional contributions during TAC. The unlabeled fractional contribution increased in MycKO-TAC versus Cont-TAC, whereas ketone and free fatty acid fractional contributions decreased. Additionally, protein posttranslational modifications by O-GlcNAc were significantly greater in Cont-TAC versus both Cont-Sham and MycKO-TAC. In conclusion, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy, which may regulate Myc-induced metabolic changes. PMID:26266538

PharmDock: a pharmacophore-based docking program

PubMed Central

2014-01-01

Background Protein-based pharmacophore models are enriched with the information of potential interactions between ligands and the protein target. We have shown in a previous study that protein-based pharmacophore models can be applied for ligand pose prediction and pose ranking. In this publication, we present a new pharmacophore-based docking program PharmDock that combines pose sampling and ranking based on optimized protein-based pharmacophore models with local optimization using an empirical scoring function. Results Tests of PharmDock on ligand pose prediction, binding affinity estimation, compound ranking and virtual screening yielded comparable or better performance to existing and widely used docking programs. The docking program comes with an easy-to-use GUI within PyMOL. Two features have been incorporated in the program suite that allow for user-defined guidance of the docking process based on previous experimental data. Docking with those features demonstrated superior performance compared to unbiased docking. Conclusion A protein pharmacophore-based docking program, PharmDock, has been made available with a PyMOL plugin. PharmDock and the PyMOL plugin are freely available from http://people.pharmacy.purdue.edu/~mlill/software/pharmdock. PMID:24739488

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins

NASA Technical Reports Server (NTRS)

Gaucher, Eric A.; Thomson, J. Michael; Burgan, Michelle F.; Benner, Steven A.

2003-01-01

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65 degrees C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria, the distribution of thermophily in derived bacterial lineages, the inferred G + C content of ancient ribosomal RNA, and the geological record combined with assumptions concerning molecular clocks. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Proteomic and Biochemical Analyses of the Cotyledon and Root of Flooding-Stressed Soybean Plants

PubMed Central

Komatsu, Setsuko; Makino, Takahiro; Yasue, Hiroshi

2013-01-01

Background Flooding significantly reduces the growth and grain yield of soybean plants. Proteomic and biochemical techniques were used to determine whether the function of cotyledon and root is altered in soybean under flooding stress. Results Two-day-old soybean plants were flooded for 2 days, after which the proteins from root and cotyledon were extracted for proteomic analysis. In response to flooding stress, the abundance of 73 and 28 proteins was significantly altered in the root and cotyledon, respectively. The accumulation of only one protein, 70 kDa heat shock protein (HSP70) (Glyma17g08020.1), increased in both organs following flooding. The ratio of protein abundance of HSP70 and biophoton emission in the cotyledon was higher than those detected in the root under flooding stress. Computed tomography and elemental analyses revealed that flooding stress decreases the number of calcium oxalate crystal the cotyledon, indicating calcium ion was elevated in the cotyledon under flooding stress. Conclusion These results suggest that calcium might play one role through HSP70 in the cotyledon under flooding stress. PMID:23799004

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

PubMed Central

Babinsky, Valerie N.; Head, Rosie A.; Cranston, Treena; Rust, Nigel; Hobbs, Maurine R.; Heath, Hunter; Thakker, Rajesh V.

2013-01-01

BACKGROUND Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2–associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2–associated mutations increased cell sensitivity. CONCLUSIONS Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.) PMID:23802516

Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs)

PubMed Central

Natale, Darren A; Shankavaram, Uma T; Galperin, Michael Y; Wolf, Yuri I; Aravind, L; Koonin, Eugene V

2000-01-01

Background: Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi. Results: A. pernix and P. abyssi proteins were assigned to COGs using the COGNITOR program; the results were verified on a case-by-case basis and augmented by additional database searches using the PSI-BLAST and TBLASTN programs. Functions were predicted for over 300 proteins from A. pernix, which could not be assigned a function using conventional methods with a conservative sequence similarity threshold, an approximately 50% increase compared to the original annotation. A. pernix shares most of the conserved core of proteins that were previously identified in the Euryarchaeota. Cluster analysis or distance matrix tree construction based on the co-occurrence of genomes in COGs showed that A. pernix forms a distinct group within the archaea, although grouping with the two species of Pyrococci, indicative of similar repertoires of conserved genes, was observed. No indication of a specific relationship between Crenarchaeota and eukaryotes was obtained in these analyses. Several proteins that are conserved in Euryarchaeota and most bacteria are unexpectedly missing in A. pernix, including the entire set of de novo purine biosynthesis enzymes, the GTPase FtsZ (a key component of the bacterial and euryarchaeal cell-division machinery), and the tRNA-specific pseudouridine synthase, previously considered universal. A. pernix is represented in 48 COGs that do not contain any euryarchaeal members. Many of these proteins are TCA cycle and electron transport chain enzymes, reflecting the aerobic lifestyle of A. pernix. Conclusions: Special-purpose databases organized on the basis of phylogenetic analysis and carefully curated with respect to known and predicted protein functions provide for a significant improvement in genome annotation. A differential genome display approach helps in a systematic investigation of common and distinct features of gene repertoires and in some cases reveals unexpected connections that may be indicative of functional similarities between phylogenetically distant organisms and of lateral gene exchange. PMID:11178258

The transcriptional activator ZNF143 is essential for normal development in zebrafish

PubMed Central

2012-01-01

Background ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. Results The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Conclusions Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development. PMID:22268977

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

PubMed

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-09-01

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design.

Assessment of potential adjuvanticity of Cry proteins.

PubMed

Joshi, Saurabh S; Barnett, Brian; Doerrer, Nancy G; Glenn, Kevin; Herman, Rod A; Herouet-Guicheney, Corinne; Hunst, Penny; Kough, John; Ladics, Gregory S; McClain, Scott; Papineni, Sabitha; Poulsen, Lars K; Rascle, Jean-Baptiste; Tao, Ai-Lin; van Ree, Ronald; Ward, Jason; Bowman, Christal C

2016-08-01

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops. Copyright © 2016 Elsevier Inc. All rights reserved.

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets

PubMed Central

2010-01-01

Background Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. Results In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Conclusions Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated. PMID:20409339

LEAping to conclusions: a computational reanalysis of late embryogenesis abundant proteins and their possible roles.

PubMed

Wise, Michael J

2003-10-29

The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and low-complexity proteins, such as the LEA proteins, pose particular challenges in determining their functions and modes of action. Rather than masking off and ignoring low-complexity domains, novel tools and tool combinations are needed which are capable of analysing such proteins in their entirety.

Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

PubMed Central

2011-01-01

Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally different groups of proteins and interactions. We conclude that it is a useful and important measure to be used in concert with gene co-expression correlation for further insights into the characteristics of proteins in the context of their interaction network. PMID:22369639

Quantity and functionality of protein fractions in chicken breast fillets affected by white striping.

PubMed

Mudalal, S; Babini, E; Cavani, C; Petracci, M

2014-08-01

Recently, white striations parallel to muscle fibers direction have been observed on the surface of chicken breast, which could be ascribed to intensive growth selection. The aim of this study was to evaluate the effect of white striping on chemical composition with special emphasis on myofibrillar and sarcoplasmic protein fractions that are relevant to the processing features of chicken breast meat. During this study, a total of 12 pectoralis major muscles from both normal and white striped fillets were used to evaluate chemical composition, protein solubility (sarcoplasmic, myofibrillar, and total protein solubility), protein quantity (sarcoplasmic, myofibrillar, and stromal proteins), water holding capacity, and protein profile by SDS-PAGE analysis. White-striped fillets exhibited a higher percentage of moisture (75.4 vs. 73.8%; P < 0.01), intramuscular fat (2.15 vs. 0.98%; P < 0.01), and collagen (1.36 vs. 1.22%; P < 0.01), and lower content of protein (18.7 vs. 22.8%; P < 0.01) and ash (1.14 vs. 1.34%; P < 0.01), in comparison with normal fillets. There was a great decline in myofibrillar (14.0 vs. 8.7%; P < 0.01) and sarcoplasmic (3.2 vs. 2.6%; P < 0.01) content and solubility as well as an increase in cooking loss (33.7 vs. 27.4%; P < 0.05) due to white striping defects. Moreover, gel electrophoresis showed that the concentration of 3 myofibrillar proteins corresponding to actin (42 kDa); LC1, slow-twitch light chain myosin (27.5 kDa); and LC3, fast-twitch light chain myosin (16 kDa), and almost all sarcoplasmic proteins were lower than normal. In conclusion, the findings of this study revealed that chicken breast meat with white striping defect had different chemical composition (more fat and less protein) and protein quality and quantity (low content of myofibrillar proteins and high content of stromal proteins) with respect to normal meat. Furthermore, white striped fillets had lower protein functionality (higher cooking loss). All the former changes indicate that white striping has great impact on quality characteristics of chicken breast meat. © Poultry Science Association Inc.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

PubMed Central

Snykers, Sarah; Vanhaecke, Tamara; De Becker, Ann; Papeleu, Peggy; Vinken, Mathieu; Van Riet, Ivan; Rogiers, Vera

2007-01-01

Background The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. Results Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. Conclusion hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells. PMID:17407549

A novel anti-aldolase C antibody specifically interacts with residues 85-102 of the protein.

PubMed

Langellotti, Simona; Romano, Maurizio; Guarnaccia, Corrado; Granata, Vincenzo; Orrù, Stefania; Zagari, Adriana; Baralle, Francisco E; Salvatore, Francesco

2014-01-01

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85-102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C's functions in the brain.

Interaction of AIM with insulin-like growth factor-binding protein-4

PubMed Central

YOU, QIANG; WU, YAN; YAO, NANNAN; SHEN, GUANNAN; ZHANG, YING; XU, LIANGGUO; LI, GUIYING; JU, CYNTHIA

2015-01-01

Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two-hybrid screening, the present study uncovered that AIM binds to insulin-like growth factor binding protein-4 (IGFBP-4). AIM interaction with IGFBP-4, as well as IGFBP-2 and -3, but not with IGFBP-1, -5 and -6, was further confirmed by co-immunoprecipitation (co-IP) using 293 cells. The binding activity and affinity between AIM and IGFBP-4 in vitro were analyzed by co-IP and biolayer interferometry. Serum depletion-induced cellular apoptosis was attenuated by insulin-like growth factor-I (IGF-I), and this effect was abrogated by IGFBP-4. Of note, in the presence of AIM, the inhibitory effect of IGFBP-4 on the anti-apoptosis function of IGF-I was attenuated, possibly through binding of AIM with IGFBP-4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP-2, -3 and -4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function. PMID:26135353

Virtual Screening and Pharmacophore Design for a Novel Theoretical Inhibitor of Macrophage Stimulating Factor as a Metastatic Agent

PubMed Central

Torktaz, Ibrahim; Mohamadhashem, Faezeh; Esmaeili, Abolghasem; Behjati, Mohaddeseh; Sharifzadeh, Sara

2013-01-01

Introduction: Metastasis is a crucial aspect of cancer. Macrophage stimulating protein (MSP) is a single chain protein and can be cleaved by serum proteases. MSP has several roles in metastasis. In this in silico study, MSP as a metastatic agent was considered as a drug target. Methods: Crystallographic structure of MSP was retrieved from protein data bank. To find a chemical inhibitor of MSP, a library of KEGG compounds was screened and 1000 shape complemented ligands were retrieved with FindSite algorithm. Molegro Virtual Docker (MVD) software was used for docking simulation of shape complemented ligands against MSP. Moldock score was used as scoring function for virtual screening and potential inhibitors with more negative binding energy were obtained. PLANS scoring function was used for revaluation of virtual screening data. Results: The top found chemical had binding affinity of -183.55 based on MolDock score and equal to -66.733 PLANTs score to MSP structure. Conclusion: Based on pharmacophore model of potential inhibitor, this study suggests that the chemical which was found in this research and its derivate can be used for subsequent laboratory studies. PMID:24163807

EasyModeller: A graphical interface to MODELLER

PubMed Central

2010-01-01

Background MODELLER is a program for automated protein Homology Modeling. It is one of the most widely used tool for homology or comparative modeling of protein three-dimensional structures, but most users find it a bit difficult to start with MODELLER as it is command line based and requires knowledge of basic Python scripting to use it efficiently. Findings The study was designed with an aim to develop of "EasyModeller" tool as a frontend graphical interface to MODELLER using Perl/Tk, which can be used as a standalone tool in windows platform with MODELLER and Python preinstalled. It helps inexperienced users to perform modeling, assessment, visualization, and optimization of protein models in a simple and straightforward way. Conclusion EasyModeller provides a graphical straight forward interface and functions as a stand-alone tool which can be used in a standard personal computer with Microsoft Windows as the operating system. PMID:20712861

PTools: an opensource molecular docking library

PubMed Central

Saladin, Adrien; Fiorucci, Sébastien; Poulain, Pierre; Prévost, Chantal; Zacharias, Martin

2009-01-01

Background Macromolecular docking is a challenging field of bioinformatics. Developing new algorithms is a slow process generally involving routine tasks that should be found in a robust library and not programmed from scratch for every new software application. Results We present an object-oriented Python/C++ library to help the development of new docking methods. This library contains low-level routines like PDB-format manipulation functions as well as high-level tools for docking and analyzing results. We also illustrate the ease of use of this library with the detailed implementation of a 3-body docking procedure. Conclusion The PTools library can handle molecules at coarse-grained or atomic resolution and allows users to rapidly develop new software. The library is already in use for protein-protein and protein-DNA docking with the ATTRACT program and for simulation analysis. This library is freely available under the GNU GPL license, together with detailed documentation. PMID:19409097

A novel stationary phase derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed protein chromatography.

PubMed

Qu, Jian-Bo; Wan, Xing-Zhong; Zhai, Yan-Qin; Zhou, Wei-Qing; Su, Zhi-Guo; Ma, Guang-Hui

2009-09-11

Using agarose coated gigaporous polystyrene microspheres as a base support, a novel anion exchanger (DEAE-AP) has been developed after functionalization with diethylaminoethyl chloride. The gigaporous structure, static adsorption behavior, and chromatographic properties of DEAE-AP medium were characterized and compared with those of commercially available resin DEAE Sepharose Fast Flow (DEAE-FF). The results implied that there existed some through pores in DEAE-AP microspheres, which effectively reduced resistance to stagnant mobile phase mass transfer by inducing convective flow of mobile phase in the gigapores of medium. As a consequence, the column packed with DEAE-AP exhibited low column backpressure, high column efficiency, high dynamic binding capacity and high protein resolution at high flow velocity up to 2600cm/h. In conclusion, all the results suggested that the gigaporous absorbent is promising for high-speed protein chromatography.

Reduced Expression of SARM in Mouse Spleen during Polymicrobial Sepsis.

PubMed

Gong, Yu; Zou, Lin; Cen, Dongzhi; Chao, Wei; Chen, Dunjin

2016-12-01

Objective Immune dysfunction, including prominent apoptosis of immune cells and decreased functioning of the remaining immune cells, plays a central role in the pathogenesis of sepsis. Sterile α and HEAT/armadillo motif-containing protein (SARM) is implicated in the regulation of immune cell apoptosis. This study aimed to elucidate SARM contributes to sepsis-induced immune cell death and immunosuppression. Methods A mouse model of polymicrobial sepsis was generated by cecum ligation and puncture (CLP). SARM gene and protein expression, caspase 3 cleavage and intracellular ATP production were measured in the mouse spleens. Results CLP-induced polymicrobial sepsis specifically attenuated both the gene and protein expression of SARM in the spleens. Moreover, the attenuation of SARM expression synchronized with splenocyte apoptosis, as evidenced by increased caspase 3 cleavage and ATP depletion. Conclusions These findings suggest that SARM is a potential regulator of sepsis-induced splenocyte apoptosis.

iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis

PubMed Central

Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

2016-01-01

Background Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. Results The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Conclusions Expression analyses of metabolism-related protein groups belonging to different functional categories and subcategories indicated that significantly upregulated proteins were related to flavonoid and starch synthesis. On the other hand, the downregulated proteins were determined to be related to nitrogen metabolism, as well as other functional categories and subcategories, including photosynthesis, redox homeostasis, tocopherol biosynthetic, and signal transduction. The results provide valuable new insights into the characterization and understanding of ACN pigment production in black rice. PMID:27415428

Evolutionary divergence of chloroplast FAD synthetase proteins

PubMed Central

2010-01-01

Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity. PMID:20955574

De novo RNA-seq and functional annotation of Ornithonyssus bacoti.

PubMed

Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li

2018-06-01

Ornithonyssus bacoti (Hirst) (Acari: Macronyssidae) is a vector and reservoir of pathogens causing serious infectious diseases, such as epidemic hemorrhagic fever, endemic typhus, tularemia, and leptospirosis. Its genome and transcriptome data are lacking in public databases. In this study, total RNA was extracted from live O. bacoti to conduct RNA-seq, functional annotation, coding domain sequence (CDS) prediction and simple sequence repeats (SSRs) detection. The results showed that 65.8 million clean reads were generated and assembled into 72,185 unigenes, of which 49.4% were annotated by seven functional databases. 23,121 unigenes were annotated and assigned to 457 species by non-redundant protein sequence database. The BLAST top-two hit species were Metaseiulus occidentalis and Ixodes scapularis. The procedure detected 12,426 SSRs, of which tri- and di-nucleotides were the most abundant types and the representative motifs were AAT/ATT and AC/GT. 26,936 CDS were predicted with a mean length of 711 bp. 87 unigenes of 30 functional genes, which are usually involved in stress responses, drug resistance, movement, metabolism and allergy, were further identified by bioinformatics methods. The unigenes putatively encoding cytochrome P450 proteins were further analyzed phylogenetically. In conclusion, this study completed the RNA-seq and functional annotation of O. bacoti successfully, which provides reliable molecular data for its future studies of gene function and molecular markers.

Multiple graph regularized protein domain ranking

PubMed Central

2012-01-01

Background Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. Results To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. Conclusion The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. PMID:23157331

Functional annotation from the genome sequence of the giant panda.

PubMed

Huo, Tong; Zhang, Yinjie; Lin, Jianping

2012-08-01

The giant panda is one of the most critically endangered species due to the fragmentation and loss of its habitat. Studying the functions of proteins in this animal, especially specific trait-related proteins, is therefore necessary to protect the species. In this work, the functions of these proteins were investigated using the genome sequence of the giant panda. Data on 21,001 proteins and their functions were stored in the Giant Panda Protein Database, in which the proteins were divided into two groups: 20,179 proteins whose functions can be predicted by GeneScan formed the known-function group, whereas 822 proteins whose functions cannot be predicted by GeneScan comprised the unknown-function group. For the known-function group, we further classified the proteins by molecular function, biological process, cellular component, and tissue specificity. For the unknown-function group, we developed a strategy in which the proteins were filtered by cross-Blast to identify panda-specific proteins under the assumption that proteins related to the panda-specific traits in the unknown-function group exist. After this filtering procedure, we identified 32 proteins (2 of which are membrane proteins) specific to the giant panda genome as compared against the dog and horse genomes. Based on their amino acid sequences, these 32 proteins were further analyzed by functional classification using SVM-Prot, motif prediction using MyHits, and interacting protein prediction using the Database of Interacting Proteins. Nineteen proteins were predicted to be zinc-binding proteins, thus affecting the activities of nucleic acids. The 32 panda-specific proteins will be further investigated by structural and functional analysis.

Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

PubMed Central

Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

2014-01-01

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

Global proteome profiling of dental cementum under experimentally-induced apposition.

PubMed

Salmon, Cristiane R; Giorgetti, Ana Paula O; Paes Leme, Adriana Franco; Domingues, Romênia R; Sallum, Enilson Antonio; Alves, Marcelo C; Kolli, Tamara N; Foster, Brian L; Nociti, Francisco H

2016-06-01

Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Wheat bran components modulate intestinal bacteria and gene expression of barrier function relevant proteins in a piglet model.

PubMed

Chen, Hong; Chen, Daiwen; Qin, Wen; Liu, Yuntao; Che, Lianqiang; Huang, Zhiqing; Luo, Yuheng; Zhang, Qing; Lin, Derong; Liu, Yaowen; Han, Guoquan; DeSmet, Stefaan; Michiels, Joris

2017-02-01

The objective of this study was to determine the impact of wheat bran and its main polysaccharides on intestinal bacteria and gene expression of intestinal barrier function relevant proteins. Thirty freshly weaned male piglets were assigned randomly to five dietary treatment groups with six piglets per group. Accordingly, five synthetic diets including a basal control diet without fiber components (CON), wheat bran diet (10% wheat bran, WB), arabinoxylan diet (AX), cellulose diet (CEL) and combined diet of arabinoxylan and cellulose (CB) were studied. The piglets were fed ad libitum for 30 d. Lower Escherichia coli (E. coli) populations in WB group and higher probiotic (Lactobacillus and Bifidobacterium) populations in groups fed diets containing arabinoxylan (WB, AX and CB) were observed and compared with CON group. Compared with CON group, the gene expressions of cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channel regulator 1 (CLCA1) and voltage-gated chloride channel 2 (CIC2) were suppressed in the WB group. And wheat bran down-regulated gene expression of pro-inflammation (TNF-α, IL-1β, IL-6) and TLRs/MyD88/NF-κB pathway compared with CON group. In conclusion, wheat bran and its main polysaccharides could change intestinal microflora and down-regulate the gene expression of intestinal barrier function relevant proteins in the distal small intestinal mucosa.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

PubMed Central

2010-01-01

Background Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs. PMID:20144196

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE PAGES

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.; ...

2017-06-27

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

Long non-coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma.

PubMed

Li, Li; Chen, Wei; Wang, Yuchuan; Tang, Luosheng; Han, Mei

2018-06-01

Retinoblastoma is the most common type of intraocular pediatric malignant tumor, which typically affects children <6 years of age. However, the underlying molecular mechanisms of retinoblastoma progression remain unclear. The aim of the present study was to investigate the function of long non-coding RNA (lncRNA) H19 in retinoblastoma clinical samples and cell lines, using reverse transcription-quantitative polymerase chain reaction, western blotting, colony formation, MTT, fluorescence activated cell sorting, cell invasion and migration, and in vivo growth assays. The results demonstrated that H19 may serve a critical oncogenic function in the progression of retinoblastoma, as lncRNA H19 levels were markedly increased in retinoblastoma cells and tissues compared with corresponding controls. In addition, patients with retinoblastoma with increased lncRNA H19 expression experienced poorer survival time compared with those with decreased lncRNA H19 levels. Knockdown of lncRNA H19 significantly suppressed retinoblastoma cell proliferation, migration and invasion in vitro and in vivo . Furthermore, lncRNA H19 expression was also associated with multiple proteins, including cyclin-dependent kinase 1, B-cell lymphoma-associated X protein, apoptosis regulator, tumor protein p53, vimentin, cadherin 13 and matrix metallopeptidase 9. In conclusion, lncRNA H19 may serve an important function in tumorigenesis and may be a potential target for therapy and prognosis in retinoblastoma.

Functional region prediction with a set of appropriate homologous sequences-an index for sequence selection by integrating structure and sequence information with spatial statistics

PubMed Central

2012-01-01

Background The detection of conserved residue clusters on a protein structure is one of the effective strategies for the prediction of functional protein regions. Various methods, such as Evolutionary Trace, have been developed based on this strategy. In such approaches, the conserved residues are identified through comparisons of homologous amino acid sequences. Therefore, the selection of homologous sequences is a critical step. It is empirically known that a certain degree of sequence divergence in the set of homologous sequences is required for the identification of conserved residues. However, the development of a method to select homologous sequences appropriate for the identification of conserved residues has not been sufficiently addressed. An objective and general method to select appropriate homologous sequences is desired for the efficient prediction of functional regions. Results We have developed a novel index to select the sequences appropriate for the identification of conserved residues, and implemented the index within our method to predict the functional regions of a protein. The implementation of the index improved the performance of the functional region prediction. The index represents the degree of conserved residue clustering on the tertiary structure of the protein. For this purpose, the structure and sequence information were integrated within the index by the application of spatial statistics. Spatial statistics is a field of statistics in which not only the attributes but also the geometrical coordinates of the data are considered simultaneously. Higher degrees of clustering generate larger index scores. We adopted the set of homologous sequences with the highest index score, under the assumption that the best prediction accuracy is obtained when the degree of clustering is the maximum. The set of sequences selected by the index led to higher functional region prediction performance than the sets of sequences selected by other sequence-based methods. Conclusions Appropriate homologous sequences are selected automatically and objectively by the index. Such sequence selection improved the performance of functional region prediction. As far as we know, this is the first approach in which spatial statistics have been applied to protein analyses. Such integration of structure and sequence information would be useful for other bioinformatics problems. PMID:22643026

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

PubMed Central

2014-01-01

Background The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Results Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. Conclusion In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus. PMID:24961398

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

PubMed Central

Tyagi, Kriti; Hossain, Mohammad Enayet; Thakur, Vandana; Aggarwal, Praveen; Malhotra, Pawan; Mohmmed, Asif; Sharma, Yagya Dutta

2016-01-01

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle. PMID:26954579

Association of innate defense proteins BPIFA1 and BPIFB1 with disease severity in COPD

PubMed Central

De Smet, Elise G; Seys, Leen JM; Verhamme, Fien M; Vanaudenaerde, Bart M; Brusselle, Guy G; Bingle, Colin D; Bracke, Ken R

2018-01-01

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal inflammatory response in the lungs caused by the inhalation of noxious particles and gases. The airway epithelium has a protective function against these harmful agents by maintaining a physical barrier and by secreting defensive proteins, such as bactericidal/permeability-increasing fold-containing (BPIF) proteins, BPIFA1 and BPIFB1. However, inconsistent data regarding BPIFA1 expression in smokers and COPD patients have been reported to date. Therefore, we investigated the expression of BPIFA1 and BPIFB1 in a large cohort of never-smokers and smokers with and without COPD, both on the messenger RNA (mRNA) level in lung tissue and on the protein level in airway epithelium. Furthermore, we examined the correlation between BPIFA1 and BPIFB1 levels, goblet cell hyperplasia, and lung function measurements. BPIFA1 and BPIFB1 mRNA expressions were significantly increased in stage III–IV COPD patients compared with stage II COPD patients and subjects without COPD. In addition, protein levels in COPD patients were significantly increased in comparison with subjects without COPD. BPIFA1 and BPIFB1 levels were inversely correlated with measurements of airflow limitation and positively correlated with goblet cell hyperplasia. In addition, by the use of immunofluorescence double staining, we demonstrated the expression of BPIFB1 in goblet cells. In conclusion, we show that BPIFA1 and BPIFB1 levels are elevated in COPD patients and correlate with disease severity. PMID:29296079

Characterization of sequences in human TWIST required for nuclear localization

PubMed Central

Singh, Shalini; Gramolini, Anthony O

2009-01-01

Background Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) 37RKRR40 and 73KRGKK77 in the human TWIST (H-TWIST) protein. Results Using site-specific mutagenesis and immunofluorescences, we observed that altered TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays. Conclusion Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4. PMID:19534813

Proteomic Analyses of Corneal Tissue Subjected to Alkali Exposure

PubMed Central

Parikh, Toral; Eisner, Natalie; Venugopalan, Praseeda; Yang, Qin; Lam, Byron L.

2011-01-01

Purpose. To determine whether exposure to alkaline chemicals results in predictable changes in corneal protein profile. To determine whether protein profile changes are indicative of severity and duration of alkali exposure. Methods. Enucleated bovine and porcine (n = 59 each) eyes were used for exposure to sodium, ammonium, and calcium hydroxide, respectively. Eyes were subjected to fluorescein staining, 5-bromo-2′-deoxy-uridine (BrdU) labeling. Excised cornea was subjected to protein extraction, spectrophotometric determination of protein amount, dynamic light scattering and SDS-PAGE profiling, mass spectrometric protein identification, and iTRAQ-labeled quantification. Select identified proteins were subjected to Western blot and immunohistochemical analyses. Results. Alkali exposure resulted in lower protein extractability from corneal tissue. Elevated aggregate formation was found with strong alkali exposure (sodium hydroxide>ammonium, calcium hydroxide), even with a short duration of exposure compared with controls. The protein yield after exposure varied as a function of postexposure time. Protein profiles changed because of alkali exposure. Concentration and strength of the alkali affected the profile change significantly. Mass spectrometry identified 15 proteins from different bands with relative quantification. Plexin D1 was identified for the first time in the cornea at a protein level that was further confirmed by Western blot and immunohistochemical analyses. Conclusions. Exposure to alkaline chemicals results in predictable and reproducible changes in corneal protein profile. Stronger alkali, longer durations, or both, of exposure resulted in lower yields and significant protein profile changes compared with controls. PMID:20861482

Towards comprehensive structural motif mining for better fold annotation in the "twilight zone" of sequence dissimilarity

PubMed Central

Jia, Yi; Huan, Jun; Buhr, Vincent; Zhang, Jintao; Carayannopoulos, Leonidas N

2009-01-01

Background Automatic identification of structure fingerprints from a group of diverse protein structures is challenging, especially for proteins whose divergent amino acid sequences may fall into the "twilight-" or "midnight-" zones where pair-wise sequence identities to known sequences fall below 25% and sequence-based functional annotations often fail. Results Here we report a novel graph database mining method and demonstrate its application to protein structure pattern identification and structure classification. The biologic motivation of our study is to recognize common structure patterns in "immunoevasins", proteins mediating virus evasion of host immune defense. Our experimental study, using both viral and non-viral proteins, demonstrates the efficiency and efficacy of the proposed method. Conclusion We present a theoretic framework, offer a practical software implementation for incorporating prior domain knowledge, such as substitution matrices as studied here, and devise an efficient algorithm to identify approximate matched frequent subgraphs. By doing so, we significantly expanded the analytical power of sophisticated data mining algorithms in dealing with large volume of complicated and noisy protein structure data. And without loss of generality, choice of appropriate compatibility matrices allows our method to be easily employed in domains where subgraph labels have some uncertainty. PMID:19208148

Proteome reference map and regulation network of neonatal rat cardiomyocyte

PubMed Central

Li, Zi-jian; Liu, Ning; Han, Qi-de; Zhang, You-yi

2011-01-01

Aim: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. Methods: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. Results: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. Conclusion: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data. PMID:21841810

Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

PubMed Central

Proença, Daniela; Faustino, Paula

2011-01-01

Background The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum. PMID:21407826

PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast

PubMed Central

2014-01-01

Background Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. Results We generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays. Conclusions Collectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription. PMID:24393166

Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies

PubMed Central

Richie, Christopher T.; Whitaker, Leslie R.; Whitaker, Keith W.; Necarsulmer, Julie; Baldwin, Heather A.; Zhang, Yajun; Fortuno, Lowella; Hinkle, Josh J.; Koivula, Pyry; Henderson, Mark J.; Sun, Wenzhi; Wang, Kai; Smith, Jeffrey C.; Pickel, Jim; Ji, Na; Hope, Bruce T.; Harvey, Brandon K.

2017-01-01

Background The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. New Method Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. Results iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. Conclusions Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo. PMID:28380331

Polycystin-1 is a Cardiomyocyte Mechanosensor That Governs L-type Ca2+ Channel Protein Stability

PubMed Central

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K.; Morales, Cyndi R.; Contreras, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J.; Somlo, Stefan; Rothermel, Beverly A.; Gillette, Thomas G.; Lavandero, Sergio; Hill, Joseph A.

2015-01-01

Background L-type calcium channel (LTCC) activity is critical to afterload-induced hypertrophic growth of the heart. However, mechanisms governing mechanical stress-induced activation of LTCC activity are obscure. Polycystin-1 (PC-1) is a G-protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results We subjected neonatal rat ventricular myocytes (NRVMs) to mechanical stretch by exposing them to hypo-osmotic (HS) medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on LTCC activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Over-expression of a C-terminal fragment of PC-1 was sufficient to trigger NRVM hypertrophy. Exposing NRVMs to HS medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 KO) and subjected them to mechanical stress in vivo (transverse aortic constriction, TAC). At baseline, PC-1 KO mice manifested decreased cardiac function relative to littermate controls, and α1C LTCC protein levels were significantly lower in PC-1 KO hearts. Whereas control mice manifested robust TAC-induced increases in cardiac mass, PC-1 KO mice showed no significant growth. Likewise, TAC-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusions PC-1 is a cardiomyocyte mechanosensor and is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. PMID:25888683

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

PubMed Central

Kumar, Rajinder; Adams, Brian; Oldenburg, Anja; Musiyenko, Alla; Barik, Sailen

2002-01-01

Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes. PMID:12057017

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

PubMed Central

Franco-Echevarría, Elsa; Sanz-Aparicio, Julia; Brearley, Charles A.; González-Rubio, Juana M.; González, Beatriz

2017-01-01

Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity. PMID:28450399

A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

DOE PAGES

Lund, C. H.; Bromley, J. R.; Stenbaek, A.; ...

2014-10-18

A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. Wemore » tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.« less

Improved protein surface comparison and application to low-resolution protein structure data

PubMed Central

2010-01-01

Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy. PMID:21172052

A draft map of the human ovarian proteome for tissue engineering and clinical applications.

PubMed

Ouni, Emna; Vertommen, Didier; Chiti, Maria Costanza; Dolmans, Marie-Madeleine; Amorim, Christiani Andrade

2018-02-23

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro and in vivo. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, western blot, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1,508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.