Evaluation of variability in high-resolution protein structures by global distance scoring.

PubMed

Anzai, Risa; Asami, Yoshiki; Inoue, Waka; Ueno, Hina; Yamada, Koya; Okada, Tetsuji

2018-01-01

Systematic analysis of the statistical and dynamical properties of proteins is critical to understanding cellular events. Extraction of biologically relevant information from a set of high-resolution structures is important because it can provide mechanistic details behind the functional properties of protein families, enabling rational comparison between families. Most of the current structural comparisons are pairwise-based, which hampers the global analysis of increasing contents in the Protein Data Bank. Additionally, pairing of protein structures introduces uncertainty with respect to reproducibility because it frequently accompanies other settings for superimposition. This study introduces intramolecular distance scoring for the global analysis of proteins, for each of which at least several high-resolution structures are available. As a pilot study, we have tested 300 human proteins and showed that the method is comprehensively used to overview advances in each protein and protein family at the atomic level. This method, together with the interpretation of the model calculations, provide new criteria for understanding specific structural variation in a protein, enabling global comparison of the variability in proteins from different species.

The protein-protein interaction network of eyestalk, Y-organ and hepatopancreas in Chinese mitten crab Eriocheir sinensis.

PubMed

Hao, Tong; Zeng, Zheng; Wang, Bin; Zhang, Yichen; Liu, Yichen; Geng, Xuyun; Sun, Jinsheng

2014-03-27

The protein-protein interaction network (PIN) is an effective information tool for understanding the complex biological processes inside the cell and solving many biological problems such as signaling pathway identification and prediction of protein functions. Eriocheir sinensis is a highly-commercial aquaculture species with an unclear proteome background which hinders the construction and development of PIN for E. sinensis. However, in recent years, the development of next-generation deep-sequencing techniques makes it possible to get high throughput data of E. sinensis tanscriptome and subsequently obtain a systematic overview of the protein-protein interaction system. In this work we sequenced the transcriptional RNA of eyestalk, Y-organ and hepatopancreas in E. sinensis and generated a PIN of E. sinensis which included 3,223 proteins and 35,787 interactions. Each protein-protein interaction in the network was scored according to the homology and genetic relationship. The signaling sub-network, representing the signal transduction pathways in E. sinensis, was extracted from the global network, which depicted a global view of the signaling systems in E. sinensis. Seven basic signal transduction pathways were identified in E. sinensis. By investigating the evolution paths of the seven pathways, we found that these pathways got mature in different evolutionary stages. Moreover, the functions of unclassified proteins and unigenes in the PIN of E. sinensis were predicted. Specifically, the functions of 549 unclassified proteins related to 864 unclassified unigenes were assigned, which respectively covered 76% and 73% of all the unclassified proteins and unigenes in the network. The PIN generated in this work is the first large-scale PIN of aquatic crustacean, thereby providing a paradigmatic blueprint of the aquatic crustacean interactome. Signaling sub-network extracted from the global PIN depicts the interaction of different signaling proteins and the evolutionary paths of the identified signal transduction pathways. Furthermore, the function assignment of unclassified proteins based on the PIN offers a new reference in protein function exploration. More importantly, the construction of the E. sinensis PIN provides necessary experience for the exploration of PINs in other aquatic crustacean species.

A General, Adaptive, Roadmap-Based Algorithm for Protein Motion Computation.

PubMed

Molloy, Kevin; Shehu, Amarda

2016-03-01

Precious information on protein function can be extracted from a detailed characterization of protein equilibrium dynamics. This remains elusive in wet and dry laboratories, as function-modulating transitions of a protein between functionally-relevant, thermodynamically-stable and meta-stable structural states often span disparate time scales. In this paper we propose a novel, robotics-inspired algorithm that circumvents time-scale challenges by drawing analogies between protein motion and robot motion. The algorithm adapts the popular roadmap-based framework in robot motion computation to handle the more complex protein conformation space and its underlying rugged energy surface. Given known structures representing stable and meta-stable states of a protein, the algorithm yields a time- and energy-prioritized list of transition paths between the structures, with each path represented as a series of conformations. The algorithm balances computational resources between a global search aimed at obtaining a global view of the network of protein conformations and their connectivity and a detailed local search focused on realizing such connections with physically-realistic models. Promising results are presented on a variety of proteins that demonstrate the general utility of the algorithm and its capability to improve the state of the art without employing system-specific insight.

Global profiling of lysine reactivity and ligandability in the human proteome

NASA Astrophysics Data System (ADS)

Hacker, Stephan M.; Backus, Keriann M.; Lazear, Michael R.; Forli, Stefano; Correia, Bruno E.; Cravatt, Benjamin F.

2017-12-01

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Global profiling of lysine reactivity and ligandability in the human proteome.

PubMed

Hacker, Stephan M; Backus, Keriann M; Lazear, Michael R; Forli, Stefano; Correia, Bruno E; Cravatt, Benjamin F

2017-12-01

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

A global trait-based approach to estimate leaf nitrogen functional allocation from observations

DOE PAGES

Ghimire, Bardan; Riley, William J.; Koven, Charles D.; ...

2017-03-28

Nitrogen is one of the most important nutrients for plant growth and a major constituent of proteins that regulate photosynthetic and respiratory processes. However, a comprehensive global analysis of nitrogen allocation in leaves for major processes with respect to different plant functional types is currently lacking. This study integrated observations from global databases with photosynthesis and respiration models to determine plant-functional-type-specific allocation patterns of leaf nitrogen for photosynthesis (Rubisco, electron transport, light absorption) and respiration (growth and maintenance), and by difference from observed total leaf nitrogen, an unexplained “residual” nitrogen pool. Based on our analysis, crops partition the largest fractionmore » of nitrogen to photosynthesis (57%) and respiration (5%) followed by herbaceous plants (44% and 4%). Tropical broadleaf evergreen trees partition the least to photosynthesis (25%) and respiration (2%) followed by needle-leaved evergreen trees (28% and 3%). In trees (especially needle-leaved evergreen and tropical broadleaf evergreen trees) a large fraction (70% and 73% respectively) of nitrogen was not explained by photosynthetic or respiratory functions. Compared to crops and herbaceous plants, this large residual pool is hypothesized to emerge from larger investments in cell wall proteins, lipids, amino acids, nucleic acid, CO2 fixation proteins (other than Rubisco), secondary compounds, and other proteins. Our estimates are different from previous studies due to differences in methodology and assumptions used in deriving nitrogen allocation estimates. Unlike previous studies, we integrate and infer nitrogen allocation estimates across multiple plant functional types, and report substantial differences in nitrogen allocation across different plant functional types. Furthermore, the resulting pattern of nitrogen allocation provides insights on mechanisms that operate at a cellular scale within leaves, and can be integrated with ecosystem models to derive emergent properties of ecosystem productivity at local, regional, and global scales.« less

A global trait-based approach to estimate leaf nitrogen functional allocation from observations

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

Ghimire, Bardan; Riley, William J.; Koven, Charles D.

Nitrogen is one of the most important nutrients for plant growth and a major constituent of proteins that regulate photosynthetic and respiratory processes. However, a comprehensive global analysis of nitrogen allocation in leaves for major processes with respect to different plant functional types is currently lacking. This study integrated observations from global databases with photosynthesis and respiration models to determine plant-functional-type-specific allocation patterns of leaf nitrogen for photosynthesis (Rubisco, electron transport, light absorption) and respiration (growth and maintenance), and by difference from observed total leaf nitrogen, an unexplained “residual” nitrogen pool. Based on our analysis, crops partition the largest fractionmore » of nitrogen to photosynthesis (57%) and respiration (5%) followed by herbaceous plants (44% and 4%). Tropical broadleaf evergreen trees partition the least to photosynthesis (25%) and respiration (2%) followed by needle-leaved evergreen trees (28% and 3%). In trees (especially needle-leaved evergreen and tropical broadleaf evergreen trees) a large fraction (70% and 73% respectively) of nitrogen was not explained by photosynthetic or respiratory functions. Compared to crops and herbaceous plants, this large residual pool is hypothesized to emerge from larger investments in cell wall proteins, lipids, amino acids, nucleic acid, CO2 fixation proteins (other than Rubisco), secondary compounds, and other proteins. Our estimates are different from previous studies due to differences in methodology and assumptions used in deriving nitrogen allocation estimates. Unlike previous studies, we integrate and infer nitrogen allocation estimates across multiple plant functional types, and report substantial differences in nitrogen allocation across different plant functional types. Furthermore, the resulting pattern of nitrogen allocation provides insights on mechanisms that operate at a cellular scale within leaves, and can be integrated with ecosystem models to derive emergent properties of ecosystem productivity at local, regional, and global scales.« less

Local-global alignment for finding 3D similarities in protein structures

DOEpatents

Zemla, Adam T [Brentwood, CA

2011-09-20

A method of finding 3D similarities in protein structures of a first molecule and a second molecule. The method comprises providing preselected information regarding the first molecule and the second molecule. Comparing the first molecule and the second molecule using Longest Continuous Segments (LCS) analysis. Comparing the first molecule and the second molecule using Global Distance Test (GDT) analysis. Comparing the first molecule and the second molecule using Local Global Alignment Scoring function (LGA_S) analysis. Verifying constructed alignment and repeating the steps to find the regions of 3D similarities in protein structures.

Global Dynamics of Proteins: Bridging Between Structure and Function

PubMed Central

Bahar, Ivet; Lezon, Timothy R.; Yang, Lee-Wei; Eyal, Eran

2010-01-01

Biomolecular systems possess unique, structure-encoded dynamic properties that underlie their biological functions. Recent studies indicate that these dynamic properties are determined to a large extent by the topology of native contacts. In recent years, elastic network models used in conjunction with normal mode analyses have proven to be useful for elucidating the collective dynamics intrinsically accessible under native state conditions, including in particular the global modes of motions that are robustly defined by the overall architecture. With increasing availability of structural data for well-studied proteins in different forms (liganded, complexed, or free), there is increasing evidence in support of the correspondence between functional changes in structures observed in experiments and the global motions predicted by these coarse-grained analyses. These observed correlations suggest that computational methods may be advantageously employed for assessing functional changes in structure and allosteric mechanisms intrinsically favored by the native fold. PMID:20192781

Global dynamics of proteins: bridging between structure and function.

PubMed

Bahar, Ivet; Lezon, Timothy R; Yang, Lee-Wei; Eyal, Eran

2010-01-01

Biomolecular systems possess unique, structure-encoded dynamic properties that underlie their biological functions. Recent studies indicate that these dynamic properties are determined to a large extent by the topology of native contacts. In recent years, elastic network models used in conjunction with normal mode analyses have proven to be useful for elucidating the collective dynamics intrinsically accessible under native state conditions, including in particular the global modes of motions that are robustly defined by the overall architecture. With increasing availability of structural data for well-studied proteins in different forms (liganded, complexed, or free), there is increasing evidence in support of the correspondence between functional changes in structures observed in experiments and the global motions predicted by these coarse-grained analyses. These observed correlations suggest that computational methods may be advantageously employed for assessing functional changes in structure and allosteric mechanisms intrinsically favored by the native fold.

Fast protein tertiary structure retrieval based on global surface shape similarity.

PubMed

Sael, Lee; Li, Bin; La, David; Fang, Yi; Ramani, Karthik; Rustamov, Raif; Kihara, Daisuke

2008-09-01

Characterization and identification of similar tertiary structure of proteins provides rich information for investigating function and evolution. The importance of structure similarity searches is increasing as structure databases continue to expand, partly due to the structural genomics projects. A crucial drawback of conventional protein structure comparison methods, which compare structures by their main-chain orientation or the spatial arrangement of secondary structure, is that a database search is too slow to be done in real-time. Here we introduce a global surface shape representation by three-dimensional (3D) Zernike descriptors, which represent a protein structure compactly as a series expansion of 3D functions. With this simplified representation, the search speed against a few thousand structures takes less than a minute. To investigate the agreement between surface representation defined by 3D Zernike descriptor and conventional main-chain based representation, a benchmark was performed against a protein classification generated by the combinatorial extension algorithm. Despite the different representation, 3D Zernike descriptor retrieved proteins of the same conformation defined by combinatorial extension in 89.6% of the cases within the top five closest structures. The real-time protein structure search by 3D Zernike descriptor will open up new possibility of large-scale global and local protein surface shape comparison. 2008 Wiley-Liss, Inc.

Determination of functional collective motions in a protein at atomic resolution using coherent neutron scattering

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

Hong, Liang; Jain, Nitin; Cheng, Xiaolin

Protein function often depends on global, collective internal motions. However, the simultaneous quantitative experimental determination of the forms, amplitudes, and time scales of these motions has remained elusive. We demonstrate that a complete description of these large-scale dynamic modes can be obtained using coherent neutron-scattering experiments on perdeuterated samples. With this approach, a microscopic relationship between the structure, dynamics, and function in a protein, cytochrome P450cam, is established. The approach developed here should be of general applicability to protein systems.

Determination of functional collective motions in a protein at atomic resolution using coherent neutron scattering

DOE PAGES

Hong, Liang; Jain, Nitin; Cheng, Xiaolin; ...

2016-10-14

Protein function often depends on global, collective internal motions. However, the simultaneous quantitative experimental determination of the forms, amplitudes, and time scales of these motions has remained elusive. We demonstrate that a complete description of these large-scale dynamic modes can be obtained using coherent neutron-scattering experiments on perdeuterated samples. With this approach, a microscopic relationship between the structure, dynamics, and function in a protein, cytochrome P450cam, is established. The approach developed here should be of general applicability to protein systems.

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

Proteomic analysis of the bacterial cell cycle

PubMed Central

Grünenfelder, Björn; Rummel, Gabriele; Vohradsky, Jiri; Röder, Daniel; Langen, Hanno; Jenal, Urs

2001-01-01

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. PMID:11287652

Global Proteomic Analysis in Trypanosomes Reveals Unique Proteins and Conserved Cellular Processes Impacted by Arginine Methylation

PubMed Central

Lott, Kaylen; Li, Jun; Fisk, John C.; Wang, Hao; Aletta, John M.; Qu, Jun; Read, Laurie K.

2013-01-01

Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of T. brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation, and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. PMID:23872088

Proteomics profiling of interactome dynamics by colocalisation analysis (COLA)† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00701e Click here for additional data file. Click here for additional data file.

PubMed Central

Sailem, Heba Z.; Kümper, Sandra; Tape, Christopher J.; McCully, Ryan R.; Paul, Angela; Anjomani-Virmouni, Sara; Jørgensen, Claus; Poulogiannis, George; Marshall, Christopher J.

2017-01-01

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein–protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision. PMID:27824369

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

PubMed

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Functional insights from proteome-wide structural modeling of Treponema pallidum subspecies pallidum, the causative agent of syphilis.

PubMed

Houston, Simon; Lithgow, Karen Vivien; Osbak, Kara Krista; Kenyon, Chris Richard; Cameron, Caroline E

2018-05-16

Syphilis continues to be a major global health threat with 11 million new infections each year, and a global burden of 36 million cases. The causative agent of syphilis, Treponema pallidum subspecies pallidum, is a highly virulent bacterium, however the molecular mechanisms underlying T. pallidum pathogenesis remain to be definitively identified. This is due to the fact that T. pallidum is currently uncultivatable, inherently fragile and thus difficult to work with, and phylogenetically distinct with no conventional virulence factor homologs found in other pathogens. In fact, approximately 30% of its predicted protein-coding genes have no known orthologs or assigned functions. Here we employed a structural bioinformatics approach using Phyre2-based tertiary structure modeling to improve our understanding of T. pallidum protein function on a proteome-wide scale. Phyre2-based tertiary structure modeling generated high-confidence predictions for 80% of the T. pallidum proteome (780/978 predicted proteins). Tertiary structure modeling also inferred the same function as primary structure-based annotations from genome sequencing pipelines for 525/605 proteins (87%), which represents 54% (525/978) of all T. pallidum proteins. Of the 175 T. pallidum proteins modeled with high confidence that were not assigned functions in the previously annotated published proteome, 167 (95%) were able to be assigned predicted functions. Twenty-one of the 175 hypothetical proteins modeled with high confidence were also predicted to exhibit significant structural similarity with proteins experimentally confirmed to be required for virulence in other pathogens. Phyre2-based structural modeling is a powerful bioinformatics tool that has provided insight into the potential structure and function of the majority of T. pallidum proteins and helped validate the primary structure-based annotation of more than 50% of all T. pallidum proteins with high confidence. This work represents the first T. pallidum proteome-wide structural modeling study and is one of few studies to apply this approach for the functional annotation of a whole proteome.

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.

A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

PubMed

Niphakis, Micah J; Lum, Kenneth M; Cognetta, Armand B; Correia, Bruno E; Ichu, Taka-Aki; Olucha, Jose; Brown, Steven J; Kundu, Soumajit; Piscitelli, Fabiana; Rosen, Hugh; Cravatt, Benjamin F

2015-06-18

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)*

PubMed Central

Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

2015-01-01

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions. PMID:25680958

The flavivirus capsid protein: Structure, function and perspectives towards drug design.

PubMed

Oliveira, Edson R A; Mohana-Borges, Ronaldo; de Alencastro, Ricardo B; Horta, Bruno A C

2017-01-02

Flaviviruses, such as dengue and zika viruses, are etiologic agents transmitted to humans mainly by arthropods and are of great epidemiological interest. The flavivirus capsid protein is a structural element required for the viral nucleocapsid assembly that presents the classical function of sheltering the viral genome. After decades of research, many reports have shown its different functionalities and influence over cell normal functioning. The subcellular distribution of this protein, which involves accumulation around lipid droplets and nuclear localization, also corroborates with its multi-functional characteristic. As flavivirus diseases are still in need of global control and in view of the possible key functionalities that the capsid protein promotes over flavivirus biology, novel considerations arise towards anti-flavivirus drug research. This review covers the main aspects concerning structural and functional features of the flavivirus C protein, ultimately, highlighting prospects in drug discovery based on this viral target. Copyright © 2016 Elsevier B.V. All rights reserved.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

PubMed

Huang, Sheng Yu; Chen, Sung Fang; Chen, Chun Hao; Huang, Hsuan Wei; Wu, Wen Guey; Sung, Wang Chou

2014-09-02

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Identification of functional candidates amongst hypothetical proteins of Treponema pallidum ssp. pallidum.

PubMed

Naqvi, Ahmad Abu Turab; Shahbaaz, Mohd; Ahmad, Faizan; Hassan, Md Imtaiyaz

2015-01-01

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.

Global Regulatory Pathways in the Alphaproteobacteria

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

none

A major goal for microbiologists in the twenty-first century is to develop an understanding of the microbial cell in all its complexity. In addition to understanding the function of individual gene products we need to focus on how the cell regulates gene expression at a global level to respond to different environmental parameters. Development of genomic technologies such as complete genome sequencing, proteomics, and global comparisons of mRNA expression patterns allows us to begin to address this issue. This proposal focuses on a number of phylogenetically related bacteria that are involved in environmentally important processes such as carbon sequestration andmore » bioremediation. Genome sequencing projects of a number of these bacteria have revealed the presence of a small family of regulatory genes found thus far only in the alpha-proteobacteria. These genes encode proteins that are related to the global regulatory protein RosR in Rhizobium etli, which is involved in determining nodulation competitiveness in this bacterium. Our goal is to examine the function of the proteins encoded by this gene family in several of the bacteria containing homologs to RosR. We will construct gene disruption mutations in a number of these bacteria and characterize the resulting mutant strains using two-dimensional gel electrophoresis and genetic and biochemical techniques. We will thus determine if the other proteins also function as global regulators of gene expression. Using proteomics methods we will identify the specific proteins whose expression varies depending on the presence or absence of the RosR homolog. Over fifty loci regulated by RosR have been identified in R. etli using transposon mutagenesis; this will serve as out benchmark to which we will compare the other regulons. We expect to identify genes regulated by RosR homologs in several bacterial species, including, but not limited to Rhodopseudomonas palustris and Sphingomonas aromaticivorans. In this way we will provide valuable information on gene regulation in this group of bacteria, expand our understanding of the evolution of global regulatory pathways, and develop methods for comparative regulon analysis among microbes.« less

Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.

PubMed

Cravatt, Benjamin F; Wright, Aaron T; Kozarich, John W

2008-01-01

Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

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

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.

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

PubMed

Dulmage, Keely A; Todor, Horia; Schmid, Amy K

2015-09-08

In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon Halobacterium salinarum is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather, H. salinarum histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that H. salinarum histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function. Copyright © 2015 Dulmage et al.

Global PROTOMAP profiling to search for biomarkers of early-recurrent hepatocellular carcinoma.

PubMed

Taoka, Masato; Morofuji, Noriaki; Yamauchi, Yoshio; Ojima, Hidenori; Kubota, Daisuke; Terukina, Goro; Nobe, Yuko; Nakayama, Hiroshi; Takahashi, Nobuhiro; Kosuge, Tomoo; Isobe, Toshiaki; Kondo, Tadashi

2014-11-07

This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent nontumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semiquantitative mass-spectrometry-based identification of in-gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8438 unique proteins from 45 708 nonredundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated or specifically proteolyzed and 41 of which were down-regulated. This data set consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and δ-catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM.

PubMed

Tuncbag, Nurcan; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2011-08-11

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.

Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei.

PubMed

Wright, Megan H; Paape, Daniel; Price, Helen P; Smith, Deborah F; Tate, Edward W

2016-06-10

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei , the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei . Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei . We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.

L-GRAAL: Lagrangian graphlet-based network aligner.

PubMed

Malod-Dognin, Noël; Pržulj, Nataša

2015-07-01

Discovering and understanding patterns in networks of protein-protein interactions (PPIs) is a central problem in systems biology. Alignments between these networks aid functional understanding as they uncover important information, such as evolutionary conserved pathways, protein complexes and functional orthologs. A few methods have been proposed for global PPI network alignments, but because of NP-completeness of underlying sub-graph isomorphism problem, producing topologically and biologically accurate alignments remains a challenge. We introduce a novel global network alignment tool, Lagrangian GRAphlet-based ALigner (L-GRAAL), which directly optimizes both the protein and the interaction functional conservations, using a novel alignment search heuristic based on integer programming and Lagrangian relaxation. We compare L-GRAAL with the state-of-the-art network aligners on the largest available PPI networks from BioGRID and observe that L-GRAAL uncovers the largest common sub-graphs between the networks, as measured by edge-correctness and symmetric sub-structures scores, which allow transferring more functional information across networks. We assess the biological quality of the protein mappings using the semantic similarity of their Gene Ontology annotations and observe that L-GRAAL best uncovers functionally conserved proteins. Furthermore, we introduce for the first time a measure of the semantic similarity of the mapped interactions and show that L-GRAAL also uncovers best functionally conserved interactions. In addition, we illustrate on the PPI networks of baker's yeast and human the ability of L-GRAAL to predict new PPIs. Finally, L-GRAAL's results are the first to show that topological information is more important than sequence information for uncovering functionally conserved interactions. L-GRAAL is coded in C++. Software is available at: http://bio-nets.doc.ic.ac.uk/L-GRAAL/. n.malod-dognin@imperial.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Mass Spectrometry Analysis of Spatial Protein Networks by Colocalization Analysis (COLA).

PubMed

Mardakheh, Faraz K

2017-01-01

A major challenge in systems biology is comprehensive mapping of protein interaction networks. Crucially, such interactions are often dynamic in nature, necessitating methods that can rapidly mine the interactome across varied conditions and treatments to reveal change in the interaction networks. Recently, we described a fast mass spectrometry-based method to reveal functional interactions in mammalian cells on a global scale, by revealing spatial colocalizations between proteins (COLA) (Mardakheh et al., Mol Biosyst 13:92-105, 2017). As protein localization and function are inherently linked, significant colocalization between two proteins is a strong indication for their functional interaction. COLA uses rapid complete subcellular fractionation, coupled with quantitative proteomics to generate a subcellular localization profile for each protein quantified by the mass spectrometer. Robust clustering is then applied to reveal significant similarities in protein localization profiles, indicative of colocalization.

Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

PubMed Central

Sael, Lee; Kihara, Daisuke

2012-01-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

PubMed

Sael, Lee; Kihara, Daisuke

2012-04-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

Global Proteomics Analysis of Protein Lysine Methylation.

PubMed

Cao, Xing-Jun; Garcia, Benjamin A

2016-11-01

Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large-scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Adaptive Local Realignment of Protein Sequences.

PubMed

DeBlasio, Dan; Kececioglu, John

2018-06-11

While mutation rates can vary markedly over the residues of a protein, multiple sequence alignment tools typically use the same values for their scoring-function parameters across a protein's entire length. We present a new approach, called adaptive local realignment, that in contrast automatically adapts to the diversity of mutation rates along protein sequences. This builds upon a recent technique known as parameter advising, which finds global parameter settings for an aligner, to now adaptively find local settings. Our approach in essence identifies local regions with low estimated accuracy, constructs a set of candidate realignments using a carefully-chosen collection of parameter settings, and replaces the region if a realignment has higher estimated accuracy. This new method of local parameter advising, when combined with prior methods for global advising, boosts alignment accuracy as much as 26% over the best default setting on hard-to-align protein benchmarks, and by 6.4% over global advising alone. Adaptive local realignment has been implemented within the Opal aligner using the Facet accuracy estimator.

Identification of Functional Candidates amongst Hypothetical Proteins of Treponema pallidum ssp. pallidum

PubMed Central

Naqvi, Ahmad Abu Turab; Shahbaaz, Mohd; Ahmad, Faizan; Hassan, Md. Imtaiyaz

2015-01-01

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions. PMID:25894582

Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

PubMed

Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

2017-10-06

Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

Improved protein model quality assessments by changing the target function.

PubMed

Uziela, Karolis; Menéndez Hurtado, David; Shu, Nanjiang; Wallner, Björn; Elofsson, Arne

2018-06-01

Protein modeling quality is an important part of protein structure prediction. We have for more than a decade developed a set of methods for this problem. We have used various types of description of the protein and different machine learning methodologies. However, common to all these methods has been the target function used for training. The target function in ProQ describes the local quality of a residue in a protein model. In all versions of ProQ the target function has been the S-score. However, other quality estimation functions also exist, which can be divided into superposition- and contact-based methods. The superposition-based methods, such as S-score, are based on a rigid body superposition of a protein model and the native structure, while the contact-based methods compare the local environment of each residue. Here, we examine the effects of retraining our latest predictor, ProQ3D, using identical inputs but different target functions. We find that the contact-based methods are easier to predict and that predictors trained on these measures provide some advantages when it comes to identifying the best model. One possible reason for this is that contact based methods are better at estimating the quality of multi-domain targets. However, training on the S-score gives the best correlation with the GDT_TS score, which is commonly used in CASP to score the global model quality. To take the advantage of both of these features we provide an updated version of ProQ3D that predicts local and global model quality estimates based on different quality estimates. © 2018 Wiley Periodicals, Inc.

GLADIATOR: a global approach for elucidating disease modules.

PubMed

Silberberg, Yael; Kupiec, Martin; Sharan, Roded

2017-05-26

Understanding the genetic basis of disease is an important challenge in biology and medicine. The observation that disease-related proteins often interact with one another has motivated numerous network-based approaches for deciphering disease mechanisms. In particular, protein-protein interaction networks were successfully used to illuminate disease modules, i.e., interacting proteins working in concert to drive a disease. The identification of these modules can further our understanding of disease mechanisms. We devised a global method for the prediction of multiple disease modules simultaneously named GLADIATOR (GLobal Approach for DIsease AssociaTed mOdule Reconstruction). GLADIATOR relies on a gold-standard disease phenotypic similarity to obtain a pan-disease view of the underlying modules. To traverse the search space of potential disease modules, we applied a simulated annealing algorithm aimed at maximizing the correlation between module similarity and the gold-standard phenotypic similarity. Importantly, this optimization is employed over hundreds of diseases simultaneously. GLADIATOR's predicted modules highly agree with current knowledge about disease-related proteins. Furthermore, the modules exhibit high coherence with respect to functional annotations and are highly enriched with known curated pathways, outperforming previous methods. Examination of the predicted proteins shared by similar diseases demonstrates the diverse role of these proteins in mediating related processes across similar diseases. Last, we provide a detailed analysis of the suggested molecular mechanism predicted by GLADIATOR for hyperinsulinism, suggesting novel proteins involved in its pathology. GLADIATOR predicts disease modules by integrating knowledge of disease-related proteins and phenotypes across multiple diseases. The predicted modules are functionally coherent and are more in line with current biological knowledge compared to modules obtained using previous disease-centric methods. The source code for GLADIATOR can be downloaded from http://www.cs.tau.ac.il/~roded/GLADIATOR.zip .

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

Clustering analysis of proteins from microbial genomes at multiple levels of resolution.

PubMed

Zaslavsky, Leonid; Ciufo, Stacy; Fedorov, Boris; Tatusova, Tatiana

2016-08-31

Microbial genomes at the National Center for Biotechnology Information (NCBI) represent a large collection of more than 35,000 assemblies. There are several complexities associated with the data: a great variation in sampling density since human pathogens are densely sampled while other bacteria are less represented; different protein families occur in annotations with different frequencies; and the quality of genome annotation varies greatly. In order to extract useful information from these sophisticated data, the analysis needs to be performed at multiple levels of phylogenomic resolution and protein similarity, with an adequate sampling strategy. Protein clustering is used to construct meaningful and stable groups of similar proteins to be used for analysis and functional annotation. Our approach is to create protein clusters at three levels. First, tight clusters in groups of closely-related genomes (species-level clades) are constructed using a combined approach that takes into account both sequence similarity and genome context. Second, clustroids of conservative in-clade clusters are organized into seed global clusters. Finally, global protein clusters are built around the the seed clusters. We propose filtering strategies that allow limiting the protein set included in global clustering. The in-clade clustering procedure, subsequent selection of clustroids and organization into seed global clusters provides a robust representation and high rate of compression. Seed protein clusters are further extended by adding related proteins. Extended seed clusters include a significant part of the data and represent all major known cell machinery. The remaining part, coming from either non-conservative (unique) or rapidly evolving proteins, from rare genomes, or resulting from low-quality annotation, does not group together well. Processing these proteins requires significant computational resources and results in a large number of questionable clusters. The developed filtering strategies allow to identify and exclude such peripheral proteins limiting the protein dataset in global clustering. Overall, the proposed methodology allows the relevant data at different levels of details to be obtained and data redundancy eliminated while keeping biologically interesting variations.

Focal Solute Trapping and Global Glymphatic Pathway Impairment in a Murine Model of Multiple Microinfarcts

PubMed Central

Wang, Minghuan; Ding, Fengfei; Deng, SaiYue; Guo, Xuequn; Wang, Wei

2017-01-01

Microinfarcts occur commonly in the aging brain as a consequence of diffuse embolic events and are associated with the development of vascular dementia and Alzheimer's disease. However, the manner in which disperse microscopic lesions reduce global cognitive function and increase the risk for Alzheimer's disease is unclear. The glymphatic system, which is a brain-wide perivascular network that supports the recirculation of CSF through the brain parenchyma, facilitates the clearance of interstitial solutes including amyloid β and tau. We investigated whether glymphatic pathway function is impaired in a murine model of multiple microinfarcts induced by intraarterial injection of cholesterol crystals. The analysis showed that multiple microinfarcts markedly impaired global influx of CSF along the glymphatic pathway. Although suppression of global glymphatic function was transient, resolving within 2 weeks of injury, CSF tracers also accumulated within tissue associated with microinfarcts. The effect of diffuse microinfarcts on global glymphatic pathway function was exacerbated in the mice aged 12 months compared with the 2- to 3-month-old mice. These findings indicate that glymphatic function is focally disrupted around microinfarcts and that the aging brain is more vulnerable to this disruption than the young brain. These observations suggest that microlesions may trap proteins and other interstitial solutes within the brain parenchyma, increasing the risk of amyloid plaque formation. SIGNIFICANCE STATEMENT Microinfarcts, small (<1 mm) ischemic lesions, are strongly associated with age-related dementia. However, how these microscopic lesions affect global cognitive function and predispose to Alzheimer's disease is unclear. The glymphatic system is a brain-wide network of channels surrounding brain blood vessels that allows CSF to exchange with interstitial fluid, clearing away cellular wastes such as amyloid β. We observed that, in mice, microinfarcts impaired global glymphatic function and solutes from the CSF became trapped in tissue associated with microinfarcts. These data suggest that small, disperse ischemic lesions can impair glymphatic function across the brain and trapping of solutes in these lesions may promote protein aggregation and neuroinflammation and eventually lead to neurodegeneration, especially in the aging brain. PMID:28188218

Focal Solute Trapping and Global Glymphatic Pathway Impairment in a Murine Model of Multiple Microinfarcts.

PubMed

Wang, Minghuan; Ding, Fengfei; Deng, SaiYue; Guo, Xuequn; Wang, Wei; Iliff, Jeffrey J; Nedergaard, Maiken

2017-03-15

Microinfarcts occur commonly in the aging brain as a consequence of diffuse embolic events and are associated with the development of vascular dementia and Alzheimer's disease. However, the manner in which disperse microscopic lesions reduce global cognitive function and increase the risk for Alzheimer's disease is unclear. The glymphatic system, which is a brain-wide perivascular network that supports the recirculation of CSF through the brain parenchyma, facilitates the clearance of interstitial solutes including amyloid β and tau. We investigated whether glymphatic pathway function is impaired in a murine model of multiple microinfarcts induced by intraarterial injection of cholesterol crystals. The analysis showed that multiple microinfarcts markedly impaired global influx of CSF along the glymphatic pathway. Although suppression of global glymphatic function was transient, resolving within 2 weeks of injury, CSF tracers also accumulated within tissue associated with microinfarcts. The effect of diffuse microinfarcts on global glymphatic pathway function was exacerbated in the mice aged 12 months compared with the 2- to 3-month-old mice. These findings indicate that glymphatic function is focally disrupted around microinfarcts and that the aging brain is more vulnerable to this disruption than the young brain. These observations suggest that microlesions may trap proteins and other interstitial solutes within the brain parenchyma, increasing the risk of amyloid plaque formation. SIGNIFICANCE STATEMENT Microinfarcts, small (<1 mm) ischemic lesions, are strongly associated with age-related dementia. However, how these microscopic lesions affect global cognitive function and predispose to Alzheimer's disease is unclear. The glymphatic system is a brain-wide network of channels surrounding brain blood vessels that allows CSF to exchange with interstitial fluid, clearing away cellular wastes such as amyloid β. We observed that, in mice, microinfarcts impaired global glymphatic function and solutes from the CSF became trapped in tissue associated with microinfarcts. These data suggest that small, disperse ischemic lesions can impair glymphatic function across the brain and trapping of solutes in these lesions may promote protein aggregation and neuroinflammation and eventually lead to neurodegeneration, especially in the aging brain. Copyright © 2017 the authors 0270-6474/17/372870-08$15.00/0.

Bromodomain proteins GTE9 and GTE11 are essential for specific BT2-mediated sugar and ABA responses in Arabidopsis thaliana.

PubMed

Misra, Anjali; McKnight, Thomas D; Mandadi, Kranthi K

2018-03-01

Global Transcription Factor Group E proteins GTE9 and GTE11 interact with BT2 to mediate ABA and sugar responses in Arabidopsis thaliana. BT2 is a BTB-domain protein that regulates responses to various hormone, stress and metabolic conditions in Arabidopsis thaliana. Loss of BT2 results in plants that are hypersensitive to inhibition of germination by abscisic acid (ABA) and sugars. Conversely, overexpression of BT2 results in resistance to ABA and sugars. Here, we report the roles of BT2-interacting partners GTE9 and GTE11, bromodomain and extraterminal-domain proteins of Global Transcription Factor Group E, in BT2-mediated responses to sugars and hormones. Loss-of-function mutants, gte9-1 and gte11-1, mimicked the bt2-1-null mutant responses; germination of all three mutants was hypersensitive to inhibition by glucose and ABA. Loss of either GTE9 or GTE11 in a BT2 over-expressing line blocked resistance to sugars and ABA, indicating that both GTE9 and GTE11 were required for BT2 function. Co-immunoprecipitation of BT2 and GTE9 suggested that these proteins physically interact in vivo, and presumably function together to mediate responses to ABA and sugar signals.

"Omics" of Food-Borne Gastroenteritis: Global Proteomic and Mutagenic Analysis of Salmonella enterica Serovar Enteritidis.

PubMed

Arunima, Aryashree; Yelamanchi, Soujanya D; Padhi, Chandrashekhar; Jaiswal, Sangeeta; Ryan, Daniel; Gupta, Bhawna; Sathe, Gajanan; Advani, Jayshree; Gowda, Harsha; Prasad, T S Keshava; Suar, Mrutyunjay

2017-10-01

Salmonella Enteritidis causes food-borne gastroenteritis by the two type three secretion systems (TTSS). TTSS-1 mediates invasion through intestinal lining, and TTSS-2 facilitates phagocytic survival. The pathogens' ability to infect effectively under TTSS-1-deficient background in host's phagocytes is poorly understood. Therefore, pathobiological understanding of TTSS-1-defective nontyphoidal Salmonellosis is highly important. We performed a comparative global proteomic analysis of the isogenic TTSS-1 mutant of Salmonella Enteritidis (M1511) and its wild-type isolate P125109. Our results showed 43 proteins were differentially expressed. Functional annotation further revealed that differentially expressed proteins belong to pathogenesis, tRNA and ncRNA metabolic processes. Three proteins, tryptophan subunit alpha chain, citrate lyase subunit alpha, and hypothetical protein 3202, were selected for in vitro analysis based on their functional annotations. Deletion mutants generated for the above proteins in the M1511 strain showed reduced intracellular survival inside macrophages in vitro. In sum, this study provides mass spectrometry-based evidence for seven hypothetical proteins, which will be subject of future investigations. Our study identifies proteins influencing virulence of Salmonella in the host. The study complements and further strengthens previously published research on proteins involved in enteropathogenesis of Salmonella and extends their role in noninvasive Salmonellosis.

Rice proteome analysis: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko; Tanaka, Naoki

2005-03-01

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

Defining the human deubiquitinating enzyme interaction landscape.

PubMed

Sowa, Mathew E; Bennett, Eric J; Gygi, Steven P; Harper, J Wade

2009-07-23

Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

Defining the Human Deubiquitinating Enzyme Interaction Landscape

PubMed Central

Sowa, Mathew E.; Bennett, Eric J.; Gygi, Steven P.; Harper, J. Wade

2009-01-01

Summary Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform, called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel non-reciprocal proteomic datasets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, sub-cellular localization and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway. PMID:19615732

A stochastic algorithm for global optimization and for best populations: A test case of side chains in proteins

PubMed Central

Glick, Meir; Rayan, Anwar; Goldblum, Amiram

2002-01-01

The problem of global optimization is pivotal in a variety of scientific fields. Here, we present a robust stochastic search method that is able to find the global minimum for a given cost function, as well as, in most cases, any number of best solutions for very large combinatorial “explosive” systems. The algorithm iteratively eliminates variable values that contribute consistently to the highest end of a cost function's spectrum of values for the full system. Values that have not been eliminated are retained for a full, exhaustive search, allowing the creation of an ordered population of best solutions, which includes the global minimum. We demonstrate the ability of the algorithm to explore the conformational space of side chains in eight proteins, with 54 to 263 residues, to reproduce a population of their low energy conformations. The 1,000 lowest energy solutions are identical in the stochastic (with two different seed numbers) and full, exhaustive searches for six of eight proteins. The others retain the lowest 141 and 213 (of 1,000) conformations, depending on the seed number, and the maximal difference between stochastic and exhaustive is only about 0.15 Kcal/mol. The energy gap between the lowest and highest of the 1,000 low-energy conformers in eight proteins is between 0.55 and 3.64 Kcal/mol. This algorithm offers real opportunities for solving problems of high complexity in structural biology and in other fields of science and technology. PMID:11792838

Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations.

PubMed

Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

2015-06-21

Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.

Effect of nutritional restriction in early pregnancy on isolated femoral artery function in mid-gestation fetal sheep

PubMed Central

Nishina, Hidenori; Green, Lucy R; McGarrigle, Hugh H G; Noakes, David E; Poston, Lucilla; Hanson, Mark A

2003-01-01

Unbalanced maternal nutrition affects fetal endocrine and cardiovascular systems, sometimes accompanied by changes in growth, although this is usually in late gestation. We determined the effect of moderate restriction for the first half of gestation of maternal dietary protein, or of total calorific intake on isolated resistance artery function of mid-gestation fetal sheep. Welsh Mountain ewes were nutritionally restricted by 30 % of the recommended nutrient intake (globally restricted) or 30 % of the recommended protein intake (protein-restricted), compared to control ewes fed 100 % of recommended nutrient intake, for ~12 days prior to conception and for the subsequent 70 days of gestation. At mid-gestation, fetal and placental weights were similar in all dietary groups. In isolated femoral arteries, the response curve to noradrenaline was reduced in protein-restricted group fetuses (P < 0.05). Maximal relaxation (P < 0.01) and sensitivity (P < 0.05) to acetylcholine were markedly reduced in protein-restricted group fetuses, and to a smaller extent in globally restricted group fetuses (response curve, P < 0.05). The dilator response (P < 0.05) and sensitivity (P < 0.05) to the α2 agonist UK14304 was lower in protein-, but not in globally restricted group fetuses. The response (P < 0.05) and sensitivity (P < 0.05) to the nitric oxide donor sodium nitroprusside were reduced in protein-restricted group fetuses compared to controls. Our data show that dietary imbalance, in particular restricted protein, of the ewe can produce blunting of endothelial-dependent and -independent relaxation in systemic arteries from the mid-gestation fetus. These changes may precede perturbed late-gestation fetal and postnatal cardiovascular control. PMID:12949230

Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4

PubMed Central

Jin, Chunhua; Cleveland, Joseph C; Ao, Lihua; Li, Jilin; Zeng, Qingchun; Fullerton, David A; Meng, Xianzhong

2014-01-01

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27. PMID:24918749

Human myocardium releases heat shock protein 27 (HSP27) after global ischemia: the proinflammatory effect of extracellular HSP27 through toll-like receptor (TLR)-2 and TLR4.

PubMed

Jin, Chunhua; Cleveland, Joseph C; Ao, Lihua; Li, Jilin; Zeng, Qingchun; Fullerton, David A; Meng, Xianzhong

2014-06-09

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.

Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

PubMed Central

Friedberg, Devorah; Midkiff, Michael; Calvo, Joseph M.

2001-01-01

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp+ and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function. PMID:11395465

Electric-field-stimulated protein mechanics

PubMed Central

Hekstra, Doeke R.; White, K. Ian; Socolich, Michael A.; Henning, Robert W.; Šrajer, Vukica; Ranganathan, Rama

2017-01-01

The internal mechanics of proteins—the coordinated motions of amino acids and the pattern of forces constraining these motions—connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function. PMID:27926732

Rice proteome database: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko

2005-09-01

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a beta-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

PubMed Central

Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

2013-01-01

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

Genomics and functional genomics in Chlamydomonas reinhardtii

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

Blaby, Ian K.; Blaby-Haas, Crysten E.

The availability of the Chlamydomonas reinhardtii nuclear genome sequence continues to enable researchers to address biological questions relevant to algae, land plants and animals in unprecedented ways. As we continue to characterize and understand biological processes in C. reinhardtii and translate that knowledge to other systems, we are faced with the realization that many genes encode proteins without a defined function. The field of functional genomics aims to close this gap between genome sequence and protein function. Transcriptomes, proteomes and phenomes can each provide layers of gene-specific functional data while supplying a global snapshot of cellular behavior under different conditions.more » Herein we present a brief history of functional genomics, the present status of the C. reinhardtii genome, how genome-wide experiments can aid in supplying protein function inferences, and provide an outlook for functional genomics in C. reinhardtii.« less

Genomics and functional genomics in Chlamydomonas reinhardtii

DOE PAGES

Blaby, Ian K.; Blaby-Haas, Crysten E.

2017-03-21

The availability of the Chlamydomonas reinhardtii nuclear genome sequence continues to enable researchers to address biological questions relevant to algae, land plants and animals in unprecedented ways. As we continue to characterize and understand biological processes in C. reinhardtii and translate that knowledge to other systems, we are faced with the realization that many genes encode proteins without a defined function. The field of functional genomics aims to close this gap between genome sequence and protein function. Transcriptomes, proteomes and phenomes can each provide layers of gene-specific functional data while supplying a global snapshot of cellular behavior under different conditions.more » Herein we present a brief history of functional genomics, the present status of the C. reinhardtii genome, how genome-wide experiments can aid in supplying protein function inferences, and provide an outlook for functional genomics in C. reinhardtii.« less

A new multi-scale method to reveal hierarchical modular structures in biological networks.

PubMed

Jiao, Qing-Ju; Huang, Yan; Shen, Hong-Bin

2016-11-15

Biological networks are effective tools for studying molecular interactions. Modular structure, in which genes or proteins may tend to be associated with functional modules or protein complexes, is a remarkable feature of biological networks. Mining modular structure from biological networks enables us to focus on a set of potentially important nodes, which provides a reliable guide to future biological experiments. The first fundamental challenge in mining modular structure from biological networks is that the quality of the observed network data is usually low owing to noise and incompleteness in the obtained networks. The second problem that poses a challenge to existing approaches to the mining of modular structure is that the organization of both functional modules and protein complexes in networks is far more complicated than was ever thought. For instance, the sizes of different modules vary considerably from each other and they often form multi-scale hierarchical structures. To solve these problems, we propose a new multi-scale protocol for mining modular structure (named ISIMB) driven by a node similarity metric, which works in an iteratively converged space to reduce the effects of the low data quality of the observed network data. The multi-scale node similarity metric couples both the local and the global topology of the network with a resolution regulator. By varying this resolution regulator to give different weightings to the local and global terms in the metric, the ISIMB method is able to fit the shape of modules and to detect them on different scales. Experiments on protein-protein interaction and genetic interaction networks show that our method can not only mine functional modules and protein complexes successfully, but can also predict functional modules from specific to general and reveal the hierarchical organization of protein complexes.

Evolution-Based Functional Decomposition of Proteins

PubMed Central

Rivoire, Olivier; Reynolds, Kimberly A.; Ranganathan, Rama

2016-01-01

The essential biological properties of proteins—folding, biochemical activities, and the capacity to adapt—arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment—a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation. PMID:27254668

Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction*

PubMed Central

Lewis, Aurélia E.; Sommer, Lilly; Arntzen, Magnus Ø.; Strahm, Yvan; Morrice, Nicholas A.; Divecha, Nullin; D'Santos, Clive S.

2011-01-01

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3–7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. PMID:21048195

NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism.

PubMed

Zorzatto, Cristiane; Machado, João Paulo B; Lopes, Kênia V G; Nascimento, Kelly J T; Pereira, Welison A; Brustolini, Otávio J B; Reis, Pedro A B; Calil, Iara P; Deguchi, Michihito; Sachetto-Martins, Gilberto; Gouveia, Bianca C; Loriato, Virgílio A P; Silva, Marcos A C; Silva, Fabyano F; Santos, Anésia A; Chory, Joanne; Fontes, Elizabeth P B

2015-04-30

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus-plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

Assessment of protein set coherence using functional annotations

PubMed Central

Chagoyen, Monica; Carazo, Jose M; Pascual-Montano, Alberto

2008-01-01

Background Analysis of large-scale experimental datasets frequently produces one or more sets of proteins that are subsequently mined for functional interpretation and validation. To this end, a number of computational methods have been devised that rely on the analysis of functional annotations. Although current methods provide valuable information (e.g. significantly enriched annotations, pairwise functional similarities), they do not specifically measure the degree of homogeneity of a protein set. Results In this work we present a method that scores the degree of functional homogeneity, or coherence, of a set of proteins on the basis of the global similarity of their functional annotations. The method uses statistical hypothesis testing to assess the significance of the set in the context of the functional space of a reference set. As such, it can be used as a first step in the validation of sets expected to be homogeneous prior to further functional interpretation. Conclusion We evaluate our method by analysing known biologically relevant sets as well as random ones. The known relevant sets comprise macromolecular complexes, cellular components and pathways described for Saccharomyces cerevisiae, which are mostly significantly coherent. Finally, we illustrate the usefulness of our approach for validating 'functional modules' obtained from computational analysis of protein-protein interaction networks. Matlab code and supplementary data are available at PMID:18937846

Opportunity for high value-added chemicals from food supply chain wastes.

PubMed

Matharu, Avtar S; de Melo, Eduardo M; Houghton, Joseph A

2016-09-01

With approximately 1.3 billion tonnes of food wasted per annum, food supply chain wastes (FSCWs) may be viewed as the contemporary Periodic Table of biobased feedstock chemicals (platform molecules) and functional materials. Herein, the global drivers and case for food waste valorisation within the context of global sustainability, sustainable development goals and the bioeconomy are discussed. The emerging potential of high value added chemicals from certain tropical FSCW is considered as these are grown in three major geographical areas: Brazil, India and China, and likely to increase in volume. FSCW in the context of biorefineries is discussed and two case studies are reported, namely: waste potato, and; orange peel waste. Interestingly, both waste feedstocks, like many others, produce proteins and with the global demand for vegetable proteins on the rise then proteins from FSCW may become a dominant area. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

A Subspace Semi-Definite programming-based Underestimation (SSDU) method for stochastic global optimization in protein docking*

PubMed Central

Nan, Feng; Moghadasi, Mohammad; Vakili, Pirooz; Vajda, Sandor; Kozakov, Dima; Ch. Paschalidis, Ioannis

2015-01-01

We propose a new stochastic global optimization method targeting protein docking problems. The method is based on finding a general convex polynomial underestimator to the binding energy function in a permissive subspace that possesses a funnel-like structure. We use Principal Component Analysis (PCA) to determine such permissive subspaces. The problem of finding the general convex polynomial underestimator is reduced into the problem of ensuring that a certain polynomial is a Sum-of-Squares (SOS), which can be done via semi-definite programming. The underestimator is then used to bias sampling of the energy function in order to recover a deep minimum. We show that the proposed method significantly improves the quality of docked conformations compared to existing methods. PMID:25914440

Patchwork structure-function analysis of the Sendai virus matrix protein.

PubMed

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

Localized structural frustration for evaluating the impact of sequence variants

PubMed Central

Kumar, Sushant; Clarke, Declan; Gerstein, Mark

2016-01-01

Population-scale sequencing is increasingly uncovering large numbers of rare single-nucleotide variants (SNVs) in coding regions of the genome. The rarity of these variants makes it challenging to evaluate their deleteriousness with conventional phenotype–genotype associations. Protein structures provide a way of addressing this challenge. Previous efforts have focused on globally quantifying the impact of SNVs on protein stability. However, local perturbations may severely impact protein functionality without strongly disrupting global stability (e.g. in relation to catalysis or allostery). Here, we describe a workflow in which localized frustration, quantifying unfavorable local interactions, is employed as a metric to investigate such effects. Using this workflow on the Protein Databank, we find that frustration produces many immediately intuitive results: for instance, disease-related SNVs create stronger changes in localized frustration than non-disease related variants, and rare SNVs tend to disrupt local interactions to a larger extent than common variants. Less obviously, we observe that somatic SNVs associated with oncogenes and tumor suppressor genes (TSGs) induce very different changes in frustration. In particular, those associated with TSGs change the frustration more in the core than the surface (by introducing loss-of-function events), whereas those associated with oncogenes manifest the opposite pattern, creating gain-of-function events. PMID:27915290

Amelioration of cognitive impairment and changes in microtubule-associated protein 2 after transient global cerebral ischemia are influenced by complex environment experience.

PubMed

Briones, Teresita L; Woods, Julie; Wadowska, Magdalena; Rogozinska, Magdalena

2006-04-03

In this study we examined whether expression of microtubule-associated protein 2 (MAP2) after transient global cerebral ischemia can be influenced by behavioral experience and if the changes are associated with functional improvement. Rats received either ischemia or sham surgery then assigned to: complex environment housing (EC) or social housing (SC) as controls for 14 days followed by water maze testing. Upregulation of MAP2 was seen in all ischemic animals with a significant overall increase evident in the EC housed rats. Behaviorally, all animals learned to perform the water maze task over time but the ischemia SC rats had the worst performance overall while all the EC housed animals demonstrated the best performance in general. Regression analysis showed that increase MAP2 expression was able to explain some of the variance in the behavioral parameters in the water maze suggesting that this cytoskeletal protein probably played a role in mediating enhanced functional outcomes.

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)*

PubMed Central

Gao, Liyan; Ge, Haitao; Huang, Xiahe; Liu, Kehui; Zhang, Yuanya; Xu, Wu; Wang, Yingchun

2015-01-01

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli. PMID:25505158

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

Illuminating structural proteins in viral "dark matter" with metaproteomics

DOE PAGES

Brum, Jennifer R.; Ignacio-Espinoza, J. Cesar; Kim, Eun -Hae; ...

2016-02-16

Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional darkmatter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore,more » four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Altogether, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.« less

Illuminating structural proteins in viral "dark matter" with metaproteomics.

PubMed

Brum, Jennifer R; Ignacio-Espinoza, J Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M; Roux, Simon; VerBerkmoes, Nathan C; Rich, Virginia I; Sullivan, Matthew B

2016-03-01

Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.

Illuminating structural proteins in viral “dark matter” with metaproteomics

PubMed Central

Brum, Jennifer R.; Ignacio-Espinoza, J. Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M.; Roux, Simon; VerBerkmoes, Nathan C.; Rich, Virginia I.; Sullivan, Matthew B.

2016-01-01

Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional “viral dark matter.” Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world’s oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter. PMID:26884177

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

PubMed Central

Usaite, Renata; Jewett, Michael C; Oliveira, Ana Paula; Yates, John R; Olsson, Lisbeth; Nielsen, Jens

2009-01-01

Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases. PMID:19888214

MetaGO: Predicting Gene Ontology of Non-homologous Proteins Through Low-Resolution Protein Structure Prediction and Protein-Protein Network Mapping.

PubMed

Zhang, Chengxin; Zheng, Wei; Freddolino, Peter L; Zhang, Yang

2018-03-10

Homology-based transferal remains the major approach to computational protein function annotations, but it becomes increasingly unreliable when the sequence identity between query and template decreases below 30%. We propose a novel pipeline, MetaGO, to deduce Gene Ontology attributes of proteins by combining sequence homology-based annotation with low-resolution structure prediction and comparison, and partner's homology-based protein-protein network mapping. The pipeline was tested on a large-scale set of 1000 non-redundant proteins from the CAFA3 experiment. Under the stringent benchmark conditions where templates with >30% sequence identity to the query are excluded, MetaGO achieves average F-measures of 0.487, 0.408, and 0.598, for Molecular Function, Biological Process, and Cellular Component, respectively, which are significantly higher than those achieved by other state-of-the-art function annotations methods. Detailed data analysis shows that the major advantage of the MetaGO lies in the new functional homolog detections from partner's homology-based network mapping and structure-based local and global structure alignments, the confidence scores of which can be optimally combined through logistic regression. These data demonstrate the power of using a hybrid model incorporating protein structure and interaction networks to deduce new functional insights beyond traditional sequence homology-based referrals, especially for proteins that lack homologous function templates. The MetaGO pipeline is available at http://zhanglab.ccmb.med.umich.edu/MetaGO/. Copyright © 2018. Published by Elsevier Ltd.

3D RNA and functional interactions from evolutionary couplings

PubMed Central

Weinreb, Caleb; Riesselman, Adam; Ingraham, John B.; Gross, Torsten; Sander, Chris; Marks, Debora S.

2016-01-01

Summary Non-coding RNAs are ubiquitous, but the discovery of new RNA gene sequences far outpaces research on their structure and functional interactions. We mine the evolutionary sequence record to derive precise information about function and structure of RNAs and RNA-protein complexes. As in protein structure prediction, we use maximum entropy global probability models of sequence co-variation to infer evolutionarily constrained nucleotide-nucleotide interactions within RNA molecules, and nucleotide-amino acid interactions in RNA-protein complexes. The predicted contacts allow all-atom blinded 3D structure prediction at good accuracy for several known RNA structures and RNA-protein complexes. For unknown structures, we predict contacts in 160 non-coding RNA families. Beyond 3D structure prediction, evolutionary couplings help identify important functional interactions, e.g., at switch points in riboswitches and at a complex nucleation site in HIV. Aided by accelerating sequence accumulation, evolutionary coupling analysis can accelerate the discovery of functional interactions and 3D structures involving RNA. PMID:27087444

Biological and functional relevance of CASP predictions.

PubMed

Liu, Tianyun; Ish-Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D; Altman, Russ B

2018-03-01

Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo-sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo-sites), and Ten sites containing important motifs, loops, or key residues with important disease-associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best-ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand-binding sites, most prediction methods have higher performance on apo-sites than holo-sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein-protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein-protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. © 2017 The Authors Proteins: Structure, Function and Bioinformatics Published by Wiley Periodicals, Inc.

Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

PubMed Central

Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

2014-01-01

We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

BiGGER: a new (soft) docking algorithm for predicting protein interactions.

PubMed

Palma, P N; Krippahl, L; Wampler, J E; Moura, J J

2000-06-01

A new computationally efficient and automated "soft docking" algorithm is described to assist the prediction of the mode of binding between two proteins, using the three-dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6-dimensional binding spaces of both molecules is systematically searched. A population of candidate protein-protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein-protein complexes of known crystallographic structures. In 22 out of 25 protein-protein complexes tested, near-native docked geometries were found with C(alpha) RMS deviations < or =4.0 A from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed.

Global analysis of translation termination in E. coli.

PubMed

Baggett, Natalie E; Zhang, Yan; Gross, Carol A

2017-03-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Using Local States To Drive the Sampling of Global Conformations in Proteins

PubMed Central

2016-01-01

Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design. PMID:26808351

Using Local States To Drive the Sampling of Global Conformations in Proteins.

PubMed

Pandini, Alessandro; Fornili, Arianna

2016-03-08

Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design.

Hierarchical Partitioning of Metazoan Protein Conservation Profiles Provides New Functional Insights

PubMed Central

Witztum, Jonathan; Persi, Erez; Horn, David; Pasmanik-Chor, Metsada; Chor, Benny

2014-01-01

The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles). We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO) analysis tool, we explore functional enrichment of the “universal proteins”, those with homologues in all 17 other species, and of the “non-universal proteins”. A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the “tree of life” (TOL consistent), as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the “life style” of the related clades. Most previous approaches for studying function and conservation are “bottom up”, studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is “top down”. We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life. PMID:24594619

Dynamic Palmitoylation and the Role of DHHC Proteins in T Cell Activation and Anergy

PubMed Central

Ladygina, Nadejda; Martin, Brent R.; Altman, Amnon

2017-01-01

Although protein S-palmitoylation was first characterized >30 years ago, and is implicated in the function, trafficking, and localization of many proteins, little is known about the regulation and physiological implications of this posttranslational modification. Palmitoylation of various signaling proteins required for TCR-induced T cell activation is also necessary for their proper function. LAT (linker for activation of T cells) is an essential scaffolding protein involved in T cell development and activation, and we found that its palmitoylation is selectively impaired in anergic T cells. The recent discovery of the DHHC family of palmitoyl acyl transferases (PATs) and the establishment of sensitive and quantitative proteomics-based methods for global analysis of the palmitoyl proteome led to significant progress in studying the biology and underlying mechanisms of cellular protein palmitoylation. We are using these approaches to explore the palmitoyl proteome in T lymphocytes and, specifically, the mechanistic basis for the impaired palmitoylation of LAT in anergic T cells. This chapter reviews the history of protein palmitoylation and its role in T cell activation, the DHHC family and new methodologies for global analysis of the palmitoyl proteome, and summarizes our recent work in this area. The new methodologies will accelerate the pace of research and provide a greatly improved mechanistic and molecular understanding of the complex process of protein palmitoylation and its regulation, and the substrate specificity of the novel DHHC family. Reversible protein palmitoylation will likely prove to be an important posttranslational mechanism that regulates cellular responses, similar to protein phosphorylation and ubiquitination. PMID:21569911

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

SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling

PubMed Central

Nie, Jia; Liu, Xiaolei; Lilley, Brendan N.; Zhang, Hai; Pan, Y. Albert; Kimball, Scot R.; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S.; Sanes, Joshua R.; Han, Xiao; Shi, Yuguang

2013-01-01

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5′ adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic β-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5′-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling. PMID:23922392

SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling.

PubMed

Nie, Jia; Liu, Xiaolei; Lilley, Brendan N; Zhang, Hai; Pan, Y Albert; Kimball, Scot R; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S; Sanes, Joshua R; Han, Xiao; Shi, Yuguang

2013-08-20

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic β-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

iDBPs: a web server for the identification of DNA binding proteins.

PubMed

Nimrod, Guy; Schushan, Maya; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2010-03-01

The iDBPs server uses the three-dimensional (3D) structure of a query protein to predict whether it binds DNA. First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence. We have trained and tested the classifier on various datasets and shown that it outperformed related methods. On a dataset that reflects the fraction of DNA binding proteins (DBPs) in a proteome, the area under the ROC curve was 0.90. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies. http://idbps.tau.ac.il/

The Physiology of Protein S-acylation

PubMed Central

Chamberlain, Luke H.; Shipston, Michael J.

2015-01-01

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

Intrinsic limits to gene regulation by global crosstalk

NASA Astrophysics Data System (ADS)

Friedlander, Tamar; Prizak, Roshan; Guet, Calin; Barton, Nicholas H.; Tkacik, Gasper

Gene activity is mediated by the specificity of binding interactions between special proteins, called transcription factors, and short regulatory sequences on the DNA, where different protein species preferentially bind different DNA targets. Limited interaction specificity may lead to crosstalk: a regulatory state in which a gene is either incorrectly activated due to spurious interactions or remains erroneously inactive. Since each protein can potentially interact with numerous DNA targets, crosstalk is inherently a global problem, yet has previously not been studied as such. We construct a theoretical framework to analyze the effects of global crosstalk on gene regulation, using statistical mechanics. We find that crosstalk in regulatory interactions puts fundamental limits on the reliability of gene regulation that are not easily mitigated by tuning proteins concentrations or by complex regulatory schemes proposed in the literature. Our results suggest that crosstalk imposes a previously unexplored global constraint on the functioning and evolution of regulatory networks, which is qualitatively distinct from the known constraints that act at the level of individual gene regulatory elements. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA Grant agreement Nr. 291734 (T.F.) and ERC Grant Nr. 250152 (N.B.).

An ABI3-interactor of conifers responds to multiple hormones.

PubMed

Zeng, Ying; Zhao, Tiehan; Kermode, Allison

2013-11-01

CnAIP2 (Callitropsis nootkatensis ABI3-Interacting Protein 2) was previously identified as a protein that interacts with the yellow-cedar ABI3 protein. CnAIP2 plays important roles during several key transitions of the plant lifecycle and acts as a global regulator with functions opposite to those of ABI3 proteins. Here we report that the CnAIP2 gene promoter is strongly upregulated by all of the major plant hormones. Young Arabidopsis seedlings expressing a chimeric CnAIP2pro-GUS construct were subjected to exogenously applied hormones; the maximum fold-enhancement of GUS activity was as high as 47-fold, and each hormone showed a distinctive cell/tissue-specific pattern of GUS induction. By far the greatest response was elicited by the synthetic auxin 2,4-D (47-fold induction); the other hormones tested stimulated GUS activities by 8- to 21-fold. The CnAIP2 promoter also responded to glucose and salt (NaCl), albeit to a lesser extent (2- to 3-fold induction). As well as acting in an antagonistic way to the global regulator ABI3, CnAIP2 appears to participate in multiple hormonal crosstalk pathways to carry out its functions.

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

PubMed

Chowdhury, Sandipan; Chanda, Baron

2010-11-02

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

Localized structural frustration for evaluating the impact of sequence variants.

PubMed

Kumar, Sushant; Clarke, Declan; Gerstein, Mark

2016-12-01

Population-scale sequencing is increasingly uncovering large numbers of rare single-nucleotide variants (SNVs) in coding regions of the genome. The rarity of these variants makes it challenging to evaluate their deleteriousness with conventional phenotype-genotype associations. Protein structures provide a way of addressing this challenge. Previous efforts have focused on globally quantifying the impact of SNVs on protein stability. However, local perturbations may severely impact protein functionality without strongly disrupting global stability (e.g. in relation to catalysis or allostery). Here, we describe a workflow in which localized frustration, quantifying unfavorable local interactions, is employed as a metric to investigate such effects. Using this workflow on the Protein Databank, we find that frustration produces many immediately intuitive results: for instance, disease-related SNVs create stronger changes in localized frustration than non-disease related variants, and rare SNVs tend to disrupt local interactions to a larger extent than common variants. Less obviously, we observe that somatic SNVs associated with oncogenes and tumor suppressor genes (TSGs) induce very different changes in frustration. In particular, those associated with TSGs change the frustration more in the core than the surface (by introducing loss-of-function events), whereas those associated with oncogenes manifest the opposite pattern, creating gain-of-function events. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

PubMed Central

Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

2016-01-01

Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR.

PubMed

Thompson, Matthew K; Ehlinger, Aaron C; Chazin, Walter J

2017-01-01

Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional dynamics of modular multidomain proteins is a major challenge confronting structural biology today because their structure is not fixed in time. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy have proven particularly useful for the analysis of the structural dynamics of modular multidomain proteins because they provide highly complementary information for characterizing the architectural landscape accessible to these proteins. SAXS provides a global snapshot of all architectural space sampled by a molecule in solution. Furthermore, SAXS is sensitive to conformational changes, organization and oligomeric states of protein assemblies, and the existence of flexibility between globular domains in multiprotein complexes. The power of NMR to characterize dynamics provides uniquely complementary information to the global snapshot of the architectural ensemble provided by SAXS because it can directly measure domain motion. In particular, NMR parameters can be used to define the diffusion of domains within modular multidomain proteins, connecting the amplitude of interdomain motion to the architectural ensemble derived from SAXS. Our laboratory has been studying the roles of modular multidomain proteins involved in human DNA replication using SAXS and NMR. Here, we present the procedure for acquiring and analyzing SAXS and NMR data, using DNA primase and replication protein A as examples. © 2017 Elsevier Inc. All rights reserved.

Sequence space and the ongoing expansion of the protein universe.

PubMed

Povolotskaya, Inna S; Kondrashov, Fyodor A

2010-06-17

The need to maintain the structural and functional integrity of an evolving protein severely restricts the repertoire of acceptable amino-acid substitutions. However, it is not known whether these restrictions impose a global limit on how far homologous protein sequences can diverge from each other. Here we explore the limits of protein evolution using sequence divergence data. We formulate a computational approach to study the rate of divergence of distant protein sequences and measure this rate for ancient proteins, those that were present in the last universal common ancestor. We show that ancient proteins are still diverging from each other, indicating an ongoing expansion of the protein sequence universe. The slow rate of this divergence is imposed by the sparseness of functional protein sequences in sequence space and the ruggedness of the protein fitness landscape: approximately 98 per cent of sites cannot accept an amino-acid substitution at any given moment but a vast majority of all sites may eventually be permitted to evolve when other, compensatory, changes occur. Thus, approximately 3.5 x 10(9) yr has not been enough to reach the limit of divergent evolution of proteins, and for most proteins the limit of sequence similarity imposed by common function may not exceed that of random sequences.

Global Survey of Protein Expression during Gonadal Sex Determination in Mice*

PubMed Central

Ewen, Katherine; Baker, Mark; Wilhelm, Dagmar; Aitken, R. John; Koopman, Peter

2009-01-01

The development of an embryo as male or female depends on differentiation of the gonads as either testes or ovaries. A number of genes are known to be important for gonadal differentiation, but our understanding of the regulatory networks underpinning sex determination remains fragmentary. To advance our understanding of sexual development beyond the transcriptome level, we performed the first global survey of the mouse gonad proteome at the time of sex determination by using two-dimensional nanoflow LC-MS/MS. The resulting data set contains a total of 1037 gene products (154 non-redundant and 883 redundant proteins) identified from 620 peptides. Functional classification and biological network construction suggested that the identified proteins primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. The data set contains potential novel regulators of gonad development and sex determination not revealed previously by transcriptomics and proteomics studies and more than 60 proteins with potential links to human disorders of sexual development. PMID:19617587

Visualizing global properties of a molecular dynamics trajectory.

PubMed

Zhou, Hao; Li, Shangyang; Makowski, Lee

2016-01-01

Molecular dynamics (MD) trajectories are very large data sets that contain substantial information about the dynamic behavior of a protein. Condensing these data into a form that can provide intuitively useful understanding of the molecular behavior during the trajectory is a substantial challenge that has received relatively little attention. Here, we introduce the sigma-r plot, a plot of the standard deviation of intermolecular distances as a function of that distance. This representation of global dynamics contains within a single, one-dimensional plot, the average range of motion between pairs of atoms within a macromolecule. Comparison of sigma-r plots calculated from 10 ns trajectories of proteins representing the four major SCOP fold classes indicates diversity of dynamic behaviors which are recognizably different among the four classes. Differences in domain structure and molecular weight also produce recognizable features in sigma-r plots, reflective of differences in global dynamics. Plots generated from trajectories with progressively increasing simulation time reflect the increased sampling of the structural ensemble as a function of time. Single amino acid replacements can give rise to changes in global dynamics detectable through comparison of sigma-r plots. Dynamic behavior of substructures can be monitored by careful choice of interatomic vectors included in the calculation. These examples provide demonstrations of the utility of the sigma-r plot to provide a simple measure of the global dynamics of a macromolecule. © 2015 Wiley Periodicals, Inc.

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

NASA Astrophysics Data System (ADS)

Soundararajan, Venky; Aravamudan, Murali

2014-12-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action - inspired by the Google page rank algorithm that unearths most ``globally connected'' websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs - up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site - encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows.

Rapid comparison of properties on protein surface

PubMed Central

Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

2008-01-01

The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM β/α barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure. PMID:18618695

Rapid comparison of properties on protein surface.

PubMed

Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

2008-10-01

The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure.

IrrE, a Global Regulator of Extreme Radiation Resistance in Deinococcus radiodurans, Enhances Salt Tolerance in Escherichia coli and Brassica napus

PubMed Central

Zhou, Zhengfu; Yan, Yongliang; Zhang, Wei; Lu, Wei; Ping, Shuzhen; Dai, Qilin; Yuan, Menglong; Feng, Bin; Hou, Xiaoguang; Zhang, Ying; Ruiqiang; Liu, Tingting; Feng, Lu; Wang, Lei; Chen, Ming; Lin, Min

2009-01-01

Background Globally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance. Methodology/Principal Findings Using plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants. Conclusions Expression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants. PMID:19204796

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.

PubMed

Hu, Pingzhao; Janga, Sarath Chandra; Babu, Mohan; Díaz-Mejía, J Javier; Butland, Gareth; Yang, Wenhong; Pogoutse, Oxana; Guo, Xinghua; Phanse, Sadhna; Wong, Peter; Chandran, Shamanta; Christopoulos, Constantine; Nazarians-Armavil, Anaies; Nasseri, Negin Karimi; Musso, Gabriel; Ali, Mehrab; Nazemof, Nazila; Eroukova, Veronika; Golshani, Ashkan; Paccanaro, Alberto; Greenblatt, Jack F; Moreno-Hagelsieb, Gabriel; Emili, Andrew

2009-04-28

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE.

PubMed

Michaux, Charlotte; Holmqvist, Erik; Vasicek, Erin; Sharan, Malvika; Barquist, Lars; Westermann, Alexander J; Gunn, John S; Vogel, Jörg

2017-06-27

The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of Salmonella enterica serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of cspC and cspE fully attenuates Salmonella in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.

A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize

PubMed Central

Musungu, Bryan; Bhatnagar, Deepak; Brown, Robert L.; Fakhoury, Ahmad M.; Geisler, Matt

2015-01-01

Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize. PMID:26089837

Palmitoylation of proteins in cancer.

PubMed

Resh, Marilyn D

2017-04-15

Post-translational modification of proteins by attachment of palmitate serves as a mechanism to regulate protein localization and function in both normal and malignant cells. Given the essential role that palmitoylation plays in cancer cell signaling, approaches that target palmitoylated proteins and palmitoyl acyltransferases (PATs) have the potential for therapeutic intervention in cancer. Highlighted here are recent advances in understanding the importance of protein palmitoylation in tumorigenic pathways. A new study has uncovered palmitoylation sites within the epidermal growth factor receptor that regulate receptor trafficking, signaling and sensitivity to tyrosine kinase inhibitors. Global data analysis from nearly 150 cancer studies reveals genomic alterations in several PATs that may account for their ability to function as tumor suppressors or oncogenes. Selective inhibitors have recently been developed that target hedgehog acyltransferase (Hhat) and Porcupine (Porcn), the acyltransferases that modify hedgehog and Wnt proteins, respectively. These inhibitors, coupled with targeted knockdown of Hhat and Porcn, reveal the essential functions of fatty acylation of secreted morphogens in a wide variety of human tumors. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Recognition of functional sites in protein structures.

PubMed

Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

2004-06-04

Recognition of regions on the surface of one protein, that are similar to a binding site of another is crucial for the prediction of molecular interactions and for functional classifications. We first describe a novel method, SiteEngine, that assumes no sequence or fold similarities and is able to recognize proteins that have similar binding sites and may perform similar functions. We achieve high efficiency and speed by introducing a low-resolution surface representation via chemically important surface points, by hashing triangles of physico-chemical properties and by application of hierarchical scoring schemes for a thorough exploration of global and local similarities. We proceed to rigorously apply this method to functional site recognition in three possible ways: first, we search a given functional site on a large set of complete protein structures. Second, a potential functional site on a protein of interest is compared with known binding sites, to recognize similar features. Third, a complete protein structure is searched for the presence of an a priori unknown functional site, similar to known sites. Our method is robust and efficient enough to allow computationally demanding applications such as the first and the third. From the biological standpoint, the first application may identify secondary binding sites of drugs that may lead to side-effects. The third application finds new potential sites on the protein that may provide targets for drug design. Each of the three applications may aid in assigning a function and in classification of binding patterns. We highlight the advantages and disadvantages of each type of search, provide examples of large-scale searches of the entire Protein Data Base and make functional predictions.

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

PubMed

D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

2009-01-01

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

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

Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Mo, Ran; Yang, Mingkun; Chen, Zhuo; Cheng, Zhongyi; Yi, Xingling; Li, Chongyang; He, Chenliu; Xiong, Qian; Chen, Hui; Wang, Qiang; Ge, Feng

2015-02-06

Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

Ligand Binding Site Detection by Local Structure Alignment and Its Performance Complementarity

PubMed Central

Lee, Hui Sun; Im, Wonpil

2013-01-01

Accurate determination of potential ligand binding sites (BS) is a key step for protein function characterization and structure-based drug design. Despite promising results of template-based BS prediction methods using global structure alignment (GSA), there is a room to improve the performance by properly incorporating local structure alignment (LSA) because BS are local structures and often similar for proteins with dissimilar global folds. We present a template-based ligand BS prediction method using G-LoSA, our LSA tool. A large benchmark set validation shows that G-LoSA predicts drug-like ligands’ positions in single-chain protein targets more precisely than TM-align, a GSA-based method, while the overall success rate of TM-align is better. G-LoSA is particularly efficient for accurate detection of local structures conserved across proteins with diverse global topologies. Recognizing the performance complementarity of G-LoSA to TM-align and a non-template geometry-based method, fpocket, a robust consensus scoring method, CMCS-BSP (Complementary Methods and Consensus Scoring for ligand Binding Site Prediction), is developed and shows improvement on prediction accuracy. The G-LoSA source code is freely available at http://im.bioinformatics.ku.edu/GLoSA. PMID:23957286

iDBPs: a web server for the identification of DNA binding proteins

PubMed Central

Nimrod, Guy; Schushan, Maya; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2010-01-01

Summary: The iDBPs server uses the three-dimensional (3D) structure of a query protein to predict whether it binds DNA. First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence. We have trained and tested the classifier on various datasets and shown that it outperformed related methods. On a dataset that reflects the fraction of DNA binding proteins (DBPs) in a proteome, the area under the ROC curve was 0.90. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies. Availability: http://idbps.tau.ac.il/ Contact: NirB@tauex.tau.ac.il Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20089514

Overview of xeroderma pigmentosum proteins architecture, mutations and post-translational modifications.

PubMed

Feltes, Bruno César; Bonatto, Diego

2015-01-01

The xeroderma pigmentosum complementation group proteins (XPs), which include XPA through XPG, play a critical role in coordinating and promoting global genome and transcription-coupled nucleotide excision repair (GG-NER and TC-NER, respectively) pathways in eukaryotic cells. GG-NER and TC-NER are both required for the repair of bulky DNA lesions, such as those induced by UV radiation. Mutations in genes that encode XPs lead to the clinical condition xeroderma pigmentosum (XP). Although the roles of XPs in the GG-NER/TC-NER subpathways have been extensively studied, complete knowledge of their three-dimensional structure is only beginning to emerge. Hence, this review aims to summarize the current knowledge of mapped mutations and other structural information on XP proteins that influence their function and protein-protein interactions. We also review the possible post-translational modifications for each protein and the impact of these modifications on XP protein functions. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome.

PubMed

Ambrosio, Alinne Batista; do Nascimento, Leandro Costa; Oliveira, Bruno V; Teixeira, Paulo José P L; Tiburcio, Ricardo A; Toledo Thomazella, Daniela P; Leme, Adriana F P; Carazzolle, Marcelo F; Vidal, Ramon O; Mieczkowski, Piotr; Meinhardt, Lyndel W; Pereira, Gonçalo A G; Cabrera, Odalys G

2013-02-11

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. 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. 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.

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

"Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

PubMed

Sinha, Indu; Karagoz, Kubra; Fogle, Rachel L; Hollenbeak, Christopher S; Zea, Arnold H; Arga, Kazim Y; Stanley, Anne E; Hawkes, Wayne C; Sinha, Raghu

2016-04-01

Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance.

Parallel Computational Protein Design.

PubMed

Zhou, Yichao; Donald, Bruce R; Zeng, Jianyang

2017-01-01

Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab (Gainza et al., Methods Enzymol 523:87, 2013) to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE (Gainza et al., PLoS Comput Biol 8:e1002335, 2012) and DEEPer (Hallen et al., Proteins 81:18-39, 2013) to also consider continuous backbone and side-chain flexibility.

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

Conformational diversity analysis reveals three functional mechanisms in proteins

PubMed Central

Fornasari, María Silvina

2017-01-01

Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call “rigid” (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions. PMID:28192432

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

PubMed

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of genetic variation on three dimensional structure and function of proteins

PubMed Central

Bhattacharya, Roshni; Rose, Peter W.; Burley, Stephen K.

2017-01-01

The Protein Data Bank (PDB; http://wwpdb.org) was established in 1971 as the first open access digital data resource in biology with seven protein structures as its initial holdings. The global PDB archive now contains more than 126,000 experimentally determined atomic level three-dimensional (3D) structures of biological macromolecules (proteins, DNA, RNA), all of which are freely accessible via the Internet. Knowledge of the 3D structure of the gene product can help in understanding its function and role in disease. Of particular interest in the PDB archive are proteins for which 3D structures of genetic variant proteins have been determined, thus revealing atomic-level structural differences caused by the variation at the DNA level. Herein, we present a systematic and qualitative analysis of such cases. We observe a wide range of structural and functional changes caused by single amino acid differences, including changes in enzyme activity, aggregation propensity, structural stability, binding, and dissociation, some in the context of large assemblies. Structural comparison of wild type and mutated proteins, when both are available, provide insights into atomic-level structural differences caused by the genetic variation. PMID:28296894

A Sinorhizobium meliloti RpoH-Regulated Gene Is Involved in Iron-Sulfur Protein Metabolism and Effective Plant Symbiosis under Intrinsic Iron Limitation.

PubMed

Sasaki, Shohei; Minamisawa, Kiwamu; Mitsui, Hisayuki

2016-09-01

In Sinorhizobium meliloti, RpoH-type sigma factors have a global impact on gene expression during heat shock and play an essential role in symbiosis with leguminous plants. Using mutational analysis of a set of genes showing highly RpoH-dependent expression during heat shock, we identified a gene indispensable for effective symbiosis. This gene, designated sufT, was located downstream of the sufBCDS homologs that specify the iron-sulfur (Fe/S) cluster assembly pathway. The identified transcription start site was preceded by an RpoH-dependent promoter consensus sequence. SufT was related to a conserved protein family of unknown molecular function, of which some members are involved in Fe/S cluster metabolism in diverse organisms. A sufT mutation decreased bacterial growth in both rich and minimal media, tolerance to stresses such as iron starvation, and activities of some Fe/S cluster-dependent enzymes. These results support the involvement of SufT in SUF (sulfur mobilization) system-mediated Fe/S protein metabolism. Furthermore, we isolated spontaneous pseudorevertants of the sufT mutant with partially recovered growth; each of them had a mutation in rirA This gene encodes a global iron regulator whose loss increases the intracellular iron content. Deletion of rirA in the original sufT mutant improved growth and restored Fe/S enzyme activities and effective symbiosis. These results suggest that enhanced iron availability compensates for the lack of SufT in the maintenance of Fe/S proteins. Although RpoH-type sigma factors of the RNA polymerase are present in diverse proteobacteria, their role as global regulators of protein homeostasis has been studied mainly in the enteric gammaproteobacterium Escherichia coli In the soil alphaproteobacterium Sinorhizobium meliloti, the rpoH mutations have a strong impact on symbiosis with leguminous plants. We found that sufT is a unique member of the S. meliloti RpoH regulon; sufT contributes to Fe/S protein metabolism and effective symbiosis under intrinsic iron limitation exerted by RirA, a global iron regulator. Our study provides insights into the RpoH regulon function in diverse proteobacteria adapted to particular ecological niches and into the mechanism of conserved Fe/S protein biogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Single proteins that serve linked functions in intracellular and extracellular microenvironments

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

Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei

2009-06-03

Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcriptsmore » that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of protein secretion (as syntaxin-2), amphoterin/high mobility group box-1 (HMGB1), which may link inflammation (as amphoterin) with regulation of gene expression (as HMGB1), and tissue transglutaminase, which affects delivery of and response to apoptotic signals by serving a related function on both sides of the plasma membrane. As it is notable that all three of these proteins have been reported to transit the plasma membrane through non-classical secretory mechanisms, we will also discuss why coordinated inside/outside functions may be found in some examples of proteins which transit the plasma membrane through non-classical mechanisms and how this relationship can be used to identify additional proteins that share these characteristics.« less

Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

PubMed Central

Mack, Korrie L.; Shorter, James

2016-01-01

Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

TaFROG encodes a Pooideae orphan protein that interacts with SnRK1 and enhances resistance to the mycotoxigenic fungus fusarium graminearum.

USDA-ARS?s Scientific Manuscript database

All genomes encode taxonomically restricted ‘orphan’ genes, most of which are of unknown function. We report the functional characterization of the orphan gene TaFROG as a component of the wheat resistance to the globally important Fusarium head blight (FHB) disease. TaFROG is taxonomically restrict...

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

Nitrosothiol signaling and protein nitrosation in cell death.

PubMed

Iyer, Anand Krishnan V; Rojanasakul, Yon; Azad, Neelam

2014-11-15

Nitric oxide, a reactive free radical, is an important signaling molecule that can lead to a plethora of cellular effects affecting homeostasis. A well-established mechanism by which NO manifests its effect on cellular functions is the post-translational chemical modification of cysteine thiols in substrate proteins by a process known as S-nitrosation. Studies that investigate regulation of cellular functions through NO have increasingly established S-nitrosation as the primary modulatory mechanism in their respective systems. There has been a substantial increase in the number of reports citing various candidate proteins undergoing S-nitrosation, which affects cell-death and -survival pathways in a number of tissues including heart, lung, brain and blood. With an exponentially growing list of proteins being identified as substrates for S-nitrosation, it is important to assimilate this information in different cell/tissue systems in order to gain an overall view of protein regulation of both individual proteins and a class of protein substrates. This will allow for broad mapping of proteins as a function of S-nitrosation, and help delineate their global effects on pathophysiological responses including cell death and survival. This information will not only provide a much better understanding of overall functional relevance of NO in the context of various disease states, it will also facilitate the generation of novel therapeutics to combat specific diseases that are driven by NO-mediated S-nitrosation. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of neuronal APL-1 expression by cholesterol starvation.

PubMed

Wiese, Mary; Antebi, Adam; Zheng, Hui

2012-01-01

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of β-amyloid plaques composed primarily of the amyloid-β peptide, a cleavage product of amyloid precursor protein (APP). While mutations in APP lead to the development of Familial Alzheimer's Disease (FAD), sporadic AD has only one clear genetic modifier: the ε4 allele of the apolipoprotein E (ApoE) gene. Cholesterol starvation in Caenorhabditis elegans leads to molting and arrest phenotypes similar to loss-of-function mutants of the APP ortholog, apl-1 (amyloid precursor-like protein 1), and lrp-1 (lipoprotein receptor-related protein 1), suggesting a potential interaction between apl-1 and cholesterol metabolism. Previously, we found that RNAi knock-down of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. Here we find the same defect is recapitulated during lrp-1 knock-down and by cholesterol starvation. A cholesterol-free diet or loss of lrp-1 directly affects APL-1 levels as both lead to loss of APL-1::GFP fluorescence in neurons. However, loss of cholesterol does not affect global transcription or protein levels as seen by qPCR and Western blot. Our results show that cholesterol and lrp-1 are involved in the regulation of synaptic transmission, similar to apl-1. Both are able to modulate APL-1 protein levels in neurons, however cholesterol changes do not affect global apl-1 transcription or APL-1 protein indicating the changes are specific to neurons. Thus, regulation of synaptic transmission and molting by LRP-1 and cholesterol may be mediated by their ability to control APL-1 neuronal protein expression.

Global analysis of translation termination in E. coli

PubMed Central

Baggett, Natalie E.

2017-01-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins. PMID:28301469

A protein-dependent side-chain rotamer library.

PubMed

Bhuyan, Md Shariful Islam; Gao, Xin

2011-12-14

Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

PubMed Central

Soundararajan, Venky; Aravamudan, Murali

2014-01-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action – inspired by the Google page rank algorithm that unearths most “globally connected” websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs – up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site – encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows. PMID:25465236

PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity.

PubMed

Feng, Baomin; Ma, Shisong; Chen, Sixue; Zhu, Ning; Zhang, Shuxin; Yu, Bin; Yu, Yu; Le, Brandon; Chen, Xuemei; Dinesh-Kumar, Savithramma P; Shan, Libo; He, Ping

2016-12-01

Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead-associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively. © 2016 The Authors.

Protein homology model refinement by large-scale energy optimization.

PubMed

Park, Hahnbeom; Ovchinnikov, Sergey; Kim, David E; DiMaio, Frank; Baker, David

2018-03-20

Proteins fold to their lowest free-energy structures, and hence the most straightforward way to increase the accuracy of a partially incorrect protein structure model is to search for the lowest-energy nearby structure. This direct approach has met with little success for two reasons: first, energy function inaccuracies can lead to false energy minima, resulting in model degradation rather than improvement; and second, even with an accurate energy function, the search problem is formidable because the energy only drops considerably in the immediate vicinity of the global minimum, and there are a very large number of degrees of freedom. Here we describe a large-scale energy optimization-based refinement method that incorporates advances in both search and energy function accuracy that can substantially improve the accuracy of low-resolution homology models. The method refined low-resolution homology models into correct folds for 50 of 84 diverse protein families and generated improved models in recent blind structure prediction experiments. Analyses of the basis for these improvements reveal contributions from both the improvements in conformational sampling techniques and the energy function.

The Global Reciprocal Reprogramming between Mycobacteriophage SWU1 and Mycobacterium Reveals the Molecular Strategy of Subversion and Promotion of Phage Infection

PubMed Central

Fan, Xiangyu; Duan, Xiangke; Tong, Yan; Huang, Qinqin; Zhou, Mingliang; Wang, Huan; Zeng, Lanying; Young, Ry F.; Xie, Jianping

2016-01-01

Bacteriophages are the viruses of bacteria, which have contributed extensively to our understanding of life and modern biology. The phage-mediated bacterial growth inhibition represents immense untapped source for novel antimicrobials. Insights into the interaction between mycobacteriophage and Mycobacterium host will inform better utilizing of mycobacteriophage. In this study, RNA sequencing technology (RNA-seq) was used to explore the global response of Mycobacterium smegmatis mc2155 at an early phase of infection with mycobacteriophage SWU1, key host metabolic processes of M. smegmatis mc2155 shut off by SWU1, and the responsible phage proteins. The results of RNA-seq were confirmed by Real-time PCR and functional assay. 1174 genes of M. smegmatis mc2155 (16.9% of the entire encoding capacity) were differentially regulated by phage infection. These genes belong to six functional categories: (i) signal transduction, (ii) cell energetics, (iii) cell wall biosynthesis, (iv) DNA, RNA, and protein biosynthesis, (v) iron uptake, (vi) central metabolism. The transcription patterns of phage SWU1 were also characterized. This study provided the first global glimpse of the reciprocal reprogramming between the mycobacteriophage and Mycobacterium host. PMID:26858712

Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks. PMID:24922508

Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks.

PubMed

Jers, Carsten; Soufi, Boumediene; Grangeasse, Christophe; Deutscher, Josef; Mijakovic, Ivan

2008-08-01

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

PubMed Central

Kaplan, Joshua M.

2008-01-01

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling. PMID:19043554

Modelling dynamics in protein crystal structures by ensemble refinement

PubMed Central

Burnley, B Tom; Afonine, Pavel V; Adams, Paul D; Gros, Piet

2012-01-01

Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships. DOI: http://dx.doi.org/10.7554/eLife.00311.001 PMID:23251785

Protein complexes, big data, machine learning and integrative proteomics: lessons learned over a decade of systematic analysis of protein interaction networks.

PubMed

Havugimana, Pierre C; Hu, Pingzhao; Emili, Andrew

2017-10-01

Elucidation of the networks of physical (functional) interactions present in cells and tissues is fundamental for understanding the molecular organization of biological systems, the mechanistic basis of essential and disease-related processes, and for functional annotation of previously uncharacterized proteins (via guilt-by-association or -correlation). After a decade in the field, we felt it timely to document our own experiences in the systematic analysis of protein interaction networks. Areas covered: Researchers worldwide have contributed innovative experimental and computational approaches that have driven the rapidly evolving field of 'functional proteomics'. These include mass spectrometry-based methods to characterize macromolecular complexes on a global-scale and sophisticated data analysis tools - most notably machine learning - that allow for the generation of high-quality protein association maps. Expert commentary: Here, we recount some key lessons learned, with an emphasis on successful workflows, and challenges, arising from our own and other groups' ongoing efforts to generate, interpret and report proteome-scale interaction networks in increasingly diverse biological contexts.

The Modular Organization of Protein Interactions in Escherichia coli

PubMed Central

Peregrín-Alvarez, José M.; Xiong, Xuejian; Su, Chong; Parkinson, John

2009-01-01

Escherichia coli serves as an excellent model for the study of fundamental cellular processes such as metabolism, signalling and gene expression. Understanding the function and organization of proteins within these processes is an important step towards a ‘systems’ view of E. coli. Integrating experimental and computational interaction data, we present a reliable network of 3,989 functional interactions between 1,941 E. coli proteins (∼45% of its proteome). These were combined with a recently generated set of 3,888 high-quality physical interactions between 918 proteins and clustered to reveal 316 discrete modules. In addition to known protein complexes (e.g., RNA and DNA polymerases), we identified modules that represent biochemical pathways (e.g., nitrate regulation and cell wall biosynthesis) as well as batteries of functionally and evolutionarily related processes. To aid the interpretation of modular relationships, several case examples are presented, including both well characterized and novel biochemical systems. Together these data provide a global view of the modular organization of the E. coli proteome and yield unique insights into structural and evolutionary relationships in bacterial networks. PMID:19798435

Unifying mechanical and thermodynamic descriptions across the thioredoxin protein family.

PubMed

Mottonen, James M; Xu, Minli; Jacobs, Donald J; Livesay, Dennis R

2009-05-15

We compare various predicted mechanical and thermodynamic properties of nine oxidized thioredoxins (TRX) using a Distance Constraint Model (DCM). The DCM is based on a nonadditive free energy decomposition scheme, where entropic contributions are determined from rigidity and flexibility of structure based on distance constraints. We perform averages over an ensemble of constraint topologies to calculate several thermodynamic and mechanical response functions that together yield quantitative stability/flexibility relationships (QSFR). Applied to the TRX protein family, QSFR metrics display a rich variety of similarities and differences. In particular, backbone flexibility is well conserved across the family, whereas cooperativity correlation describing mechanical and thermodynamic couplings between the residue pairs exhibit distinctive features that readily standout. The diversity in predicted QSFR metrics that describe cooperativity correlation between pairs of residues is largely explained by a global flexibility order parameter describing the amount of intrinsic flexibility within the protein. A free energy landscape is calculated as a function of the flexibility order parameter, and key values are determined where the native-state, transition-state, and unfolded-state are located. Another key value identifies a mechanical transition where the global nature of the protein changes from flexible to rigid. The key values of the flexibility order parameter help characterize how mechanical and thermodynamic response is linked. Variation in QSFR metrics and key characteristics of global flexibility are related to the native state X-ray crystal structure primarily through the hydrogen bond network. Furthermore, comparison of three TRX redox pairs reveals differences in thermodynamic response (i.e., relative melting point) and mechanical properties (i.e., backbone flexibility and cooperativity correlation) that are consistent with experimental data on thermal stabilities and NMR dynamical profiles. The results taken together demonstrate that small-scale structural variations are amplified into discernible global differences by propagating mechanical couplings through the H-bond network.

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity.

PubMed

Voloshchuk, Natalya; Zhu, Anita Y; Snydacker, David; Montclare, Jin Kim

2009-09-15

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.

Proton transfer and protein quake in photoreceptor activation

NASA Astrophysics Data System (ADS)

Xie, Aihua

2002-03-01

Proteins are able to perform an enormous variety of functions, while using only a limited number of underlying processes. One of these is proton transfer, found in a range of receptors and enzymes. It is conceivable that proton transfer is essential in biological energy transduction, but it is less evident how proton transfer is employed in receptor activation during biological signal transduction. An important question regarding receptor activation is how a localized event of detecting a stimulus at the active site drives global conformational changes involving protein surface for signal relay. We will present structural, kinetic and energetic studies on the activation mechanism of a prototype PAS domain photoreceptor, photoactive yellow protein (PYP). Our data reveal that the putative signaling state of PYP upon absorption of a blue photon is formed during a large-amplitude protein quake triggered by the formation of a new buried charge in a hydrophobic pocket at the active site of PYP via intramolecular proton transfer. This mechanism for protein quakes driven by proton transfer and electrostatic interactions may play roles during the functioning of other receptor proteins and non-receptor proteins that require large conformational changes.

Multiple-Localization and Hub Proteins

PubMed Central

Ota, Motonori; Gonja, Hideki; Koike, Ryotaro; Fukuchi, Satoshi

2016-01-01

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing. PMID:27285823

Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

PubMed

Bershtein, Shimon; Serohijos, Adrian W R; Bhattacharyya, Sanchari; Manhart, Michael; Choi, Jeong-Mo; Mu, Wanmeng; Zhou, Jingwen; Shakhnovich, Eugene I

2015-10-01

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.

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

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

Gel-Based Hippocampal Proteomic Analysis 2 Weeks following Traumatic Brain Injury to Immature Rats Using Controlled Cortical Impact

PubMed Central

Kochanek, Ashley R.; Kline, Anthony E.; Gao, Wei-Min; Chadha, Mandeep; Lai, Yichen; Clark, Robert S.B.; Dixon, C. Edward; Jenkins, Larry W.

2009-01-01

Traumatic brain injury (TBI) to postnatal day 17 rats has been shown to produce acute changes in hippocampal global protein levels and spatial learning and memory deficits. The purpose of the present study was to analyze global hippocampal protein changes 2 weeks after a moderate ipsilateral controlled cortical impact in postnatal day 17 rats using 2-dimensional difference gel electrophoresis and mass spectrometry. Paired sham and ipsilateral injured hippocampal lysates were independently labeled with different fluorescent cyanine dyes and coseparated within the same immobilized pH gradient strips and slab gel based on isoelectric point and molecular mass. Significant changes in key proteins involved in glial and neuronal stress, oxidative metabolism, calcium uptake and neurotransmitter function were found 2 weeks after injury, and their potential roles in hippocampal plasticity and cognitive dysfunction were discussed. PMID:16943664

Serial interactome capture of the human cell nucleus.

PubMed

Conrad, Thomas; Albrecht, Anne-Susann; de Melo Costa, Veronica Rodrigues; Sauer, Sascha; Meierhofer, David; Ørom, Ulf Andersson

2016-04-04

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.

Identification and characterization of the grape WRKY family.

PubMed

Zhang, Ying; Feng, Jian Can

2014-01-01

WRKY transcription factors have functions in plant growth and development and in response to biotic and abiotic stresses. Many studies have focused on functional identification of WRKY transcription factors, but little is known about the molecular phylogeny or global expression patterns of the complete WRKY family. In this study, we identified 80 WRKY proteins encoded in the grape genome. Based on the structural features of these proteins, the grape WRKY genes were classified into three groups (groups 1-3). Analysis of WRKY genes expression profiles indicated that 28 WRKY genes were differentially expressed in response to biotic stress caused by grape whiterot and/or salicylic acid (SA). In that 16 WRKY genes upregulated both by whiterot pathogenic bacteria and SA. The results indicated that 16 WRKY proteins participated in SA-dependent defense signal pathway. This study provides a basis for cloning genes with specific functions from grape.

Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

PubMed Central

Woo, Hye Ryun; Dittmer, Travis A.; Richards, Eric J.

2008-01-01

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing. PMID:18704160

Integrative network alignment reveals large regions of global network similarity in yeast and human.

PubMed

Kuchaiev, Oleksii; Przulj, Natasa

2011-05-15

High-throughput methods for detecting molecular interactions have produced large sets of biological network data with much more yet to come. Analogous to sequence alignment, efficient and reliable network alignment methods are expected to improve our understanding of biological systems. Unlike sequence alignment, network alignment is computationally intractable. Hence, devising efficient network alignment heuristics is currently a foremost challenge in computational biology. We introduce a novel network alignment algorithm, called Matching-based Integrative GRAph ALigner (MI-GRAAL), which can integrate any number and type of similarity measures between network nodes (e.g. proteins), including, but not limited to, any topological network similarity measure, sequence similarity, functional similarity and structural similarity. Hence, we resolve the ties in similarity measures and find a combination of similarity measures yielding the largest contiguous (i.e. connected) and biologically sound alignments. MI-GRAAL exposes the largest functional, connected regions of protein-protein interaction (PPI) network similarity to date: surprisingly, it reveals that 77.7% of proteins in the baker's yeast high-confidence PPI network participate in such a subnetwork that is fully contained in the human high-confidence PPI network. This is the first demonstration that species as diverse as yeast and human contain so large, continuous regions of global network similarity. We apply MI-GRAAL's alignments to predict functions of un-annotated proteins in yeast, human and bacteria validating our predictions in the literature. Furthermore, using network alignment scores for PPI networks of different herpes viruses, we reconstruct their phylogenetic relationship. This is the first time that phylogeny is exactly reconstructed from purely topological alignments of PPI networks. Supplementary files and MI-GRAAL executables: http://bio-nets.doc.ic.ac.uk/MI-GRAAL/.

Protein Loop Structure Prediction Using Conformational Space Annealing.

PubMed

Heo, Seungryong; Lee, Juyong; Joo, Keehyoung; Shin, Hang-Cheol; Lee, Jooyoung

2017-05-22

We have developed a protein loop structure prediction method by combining a new energy function, which we call E PLM (energy for protein loop modeling), with the conformational space annealing (CSA) global optimization algorithm. The energy function includes stereochemistry, dynamic fragment assembly, distance-scaled finite ideal gas reference (DFIRE), and generalized orientation- and distance-dependent terms. For the conformational search of loop structures, we used the CSA algorithm, which has been quite successful in dealing with various hard global optimization problems. We assessed the performance of E PLM with two widely used loop-decoy sets, Jacobson and RAPPER, and compared the results against the DFIRE potential. The accuracy of model selection from a pool of loop decoys as well as de novo loop modeling starting from randomly generated structures was examined separately. For the selection of a nativelike structure from a decoy set, E PLM was more accurate than DFIRE in the case of the Jacobson set and had similar accuracy in the case of the RAPPER set. In terms of sampling more nativelike loop structures, E PLM outperformed E DFIRE for both decoy sets. This new approach equipped with E PLM and CSA can serve as the state-of-the-art de novo loop modeling method.

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Processing of Cholinesterase-like α/β-Hydrolase Fold Proteins: Alterations Associated with Congenital Disorders

PubMed Central

De Jaco, Antonella; Comoletti, Davide; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2016-01-01

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β-hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold. PMID:21933121

Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E.; Schwartz, Stanley A.

2010-01-01

Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse. PMID:19811410

Biological and functional relevance of CASP predictions

PubMed Central

Liu, Tianyun; Ish‐Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D.

2017-01-01

Abstract Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo‐sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo‐sites), and Ten sites containing important motifs, loops, or key residues with important disease‐associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best‐ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand‐binding sites, most prediction methods have higher performance on apo‐sites than holo‐sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein‐protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein‐protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. PMID:28975675

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence

PubMed Central

Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

2016-01-01

Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain duplications, the reconstruction of the history of protein architectures, and the estimation of protein domain age. Website and software: http://www.lcqb.upmc.fr/CLADE. PMID:27472895

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line.

PubMed

Shirata, Noriko; Ikeda, Motoko; Kobayashi, Michihiro

2010-03-15

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV. Copyright 2009 Elsevier Inc. All rights reserved.

Multiple graph regularized protein domain ranking.

PubMed

Wang, Jim Jing-Yan; Bensmail, Halima; Gao, Xin

2012-11-19

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. 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. 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.

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

Building toy models of proteins using coevolutionary information

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Raghunathan, Mohit; Onuchic, Jose

2015-03-01

Recent developments in global statistical methodologies have advanced the analysis of large collections of protein sequences for coevolutionary information. Coevolution between amino acids in a protein arises from compensatory mutations that are needed to maintain the stability or function of a protein over the course of evolution. This gives rise to quantifiable correlations between amino acid positions within the multiple sequence alignment of a protein family. Here, we use Direct Coupling Analysis (DCA) to infer a Potts model Hamiltonian governing the correlated mutations in a protein family to obtain the sequence-dependent interaction energies of a toy protein model. We demonstrate that this methodology predicts residue-residue interaction energies that are consistent with experimental mutational changes in protein stabilities as well as other computational methodologies. Furthermore, we demonstrate with several examples that DCA could be used to construct a structure-based model that quantitatively agrees with experimental data on folding mechanisms. This work serves as a potential framework for generating models of proteins that are enriched by evolutionary data that can potentially be used to engineer key functional motions and interactions in protein systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1427654).

Translatomics combined with transcriptomics and proteomics reveals novel functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).

PubMed

Neuhaus, Klaus; Landstorfer, Richard; Fellner, Lea; Simon, Svenja; Schafferhans, Andrea; Goldberg, Tatyana; Marx, Harald; Ozoline, Olga N; Rost, Burkhard; Kuster, Bernhard; Keim, Daniel A; Scherer, Siegfried

2016-02-24

Genomes of E. coli, including that of the human pathogen Escherichia coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which, apparently, have escaped annotation due to their small size and non-essential function. To find such genes, global gene expression of EHEC EDL933 was examined, using strand-specific RNAseq (transcriptome), ribosomal footprinting (translatome) and mass spectrometry (proteome). Using the above methods, 72 short, non-annotated protein-coding genes were detected. All of these showed signals in the ribosomal footprinting assay indicating mRNA translation. Seven were verified by mass spectrometry. Fifty-seven genes are annotated in other enterobacteriaceae, mainly as hypothetical genes; the remaining 15 genes constitute novel discoveries. In addition, protein structure and function were predicted computationally and compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. Based on this comparison, 61 of the 72 novel proteins exhibit predicted structural and functional features similar to those of annotated proteins. Many of the novel genes show differential transcription when grown under eleven diverse growth conditions suggesting environmental regulation. Three genes were found to confer a phenotype in previous studies, e.g., decreased cattle colonization. These findings demonstrate that ribosomal footprinting can be used to detect novel protein coding genes, contributing to the growing body of evidence that hypothetical genes are not annotation artifacts and opening an additional way to study their functionality. All 72 genes are taxonomically restricted and, therefore, appear to have evolved relatively recently de novo.

Structure and dynamics of Ebola virus matrix protein VP40 by a coarse-grained Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Pandey, Ras; Farmer, Barry

Ebola virus matrix protein VP40 (consisting of 326 residues) plays a critical role in viral assembly and its functions such as regulation of viral transcription, packaging, and budding of mature virions into the plasma membrane of infected cells. How does the protein VP40 go through structural evolution during the viral life cycle remains an open question? Using a coarse-grained Monte Carlo simulation we investigate the structural evolution of VP40 as a function of temperature with the input of a knowledge-based residue-residue interaction. A number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) are analyzed with our large-scale simulations. Our preliminary data show that the structure of the protein evolves through different state with well-defined morphologies which can be identified and quantified via a detailed analysis of structure factor.

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

PubMed Central

Hernández-Ramírez, Laura C.; Morgan, Rhodri M.L.; Barry, Sayka; D’Acquisto, Fulvio; Prodromou, Chrisostomos; Korbonits, Márta

2018-01-01

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP. PMID:29507682

Allostery in the ferredoxin protein motif does not involve a conformational switch.

PubMed

Nechushtai, Rachel; Lammert, Heiko; Michaeli, Dorit; Eisenberg-Domovich, Yael; Zuris, John A; Luca, Maria A; Capraro, Dominique T; Fish, Alex; Shimshon, Odelia; Roy, Melinda; Schug, Alexander; Whitford, Paul C; Livnah, Oded; Onuchic, José N; Jennings, Patricia A

2011-02-08

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.

Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

PubMed

Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

2015-04-01

Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein-protein structure prediction by scoring molecular dynamics trajectories of putative poses.

PubMed

Sarti, Edoardo; Gladich, Ivan; Zamuner, Stefano; Correia, Bruno E; Laio, Alessandro

2016-09-01

The prediction of protein-protein interactions and their structural configuration remains a largely unsolved problem. Most of the algorithms aimed at finding the native conformation of a protein complex starting from the structure of its monomers are based on searching the structure corresponding to the global minimum of a suitable scoring function. However, protein complexes are often highly flexible, with mobile side chains and transient contacts due to thermal fluctuations. Flexibility can be neglected if one aims at finding quickly the approximate structure of the native complex, but may play a role in structure refinement, and in discriminating solutions characterized by similar scores. We here benchmark the capability of some state-of-the-art scoring functions (BACH-SixthSense, PIE/PISA and Rosetta) in discriminating finite-temperature ensembles of structures corresponding to the native state and to non-native configurations. We produce the ensembles by running thousands of molecular dynamics simulations in explicit solvent starting from poses generated by rigid docking and optimized in vacuum. We find that while Rosetta outperformed the other two scoring functions in scoring the structures in vacuum, BACH-SixthSense and PIE/PISA perform better in distinguishing near-native ensembles of structures generated by molecular dynamics in explicit solvent. Proteins 2016; 84:1312-1320. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

PubMed

Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

2013-07-01

The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

Structural Variation of Alpha-synuclein with Temperature by a Coarse-grained Approach with Knowledge-based Interactions (Postprint)

DTIC Science & Technology

2015-07-01

the radius of gyration in detail as a function FIG. 5. Variation of the root mean square (RMS) displacement of the center of mass of the protein with...depends on the temperature. The global motion can be examined by analyzing the variation of the root mean square displacement (RMS) of the center of...and global physical quantities during the course of simula- tion, including the energy of each residue, its mobility, mean square displacement of the

Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station

PubMed Central

Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

2014-01-01

The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected. PMID:24618597

Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

PubMed

Mazars, Christian; Brière, Christian; Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

2014-01-01

The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.

Reconstructing targetable pathways in lung cancer by integrating diverse omics data

PubMed Central

Balbin, O. Alejandro; Prensner, John R.; Sahu, Anirban; Yocum, Anastasia; Shankar, Sunita; Malik, Rohit; Fermin, Damian; Dhanasekaran, Saravana M.; Chandler, Benjamin; Thomas, Dafydd; Beer, David G.; Cao, Xuhong; Nesvizhskii, Alexey I.; Chinnaiyan, Arul M.

2014-01-01

Global ‘multi-omics’ profiling of cancer cells harbours the potential for characterizing the signaling networks associated with specific oncogenes. Here we profile the transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate data sets. We first define an ‘abundance-score’ combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centered on KRAS and MET, LCK and PAK1 and b-Catenin. We validate activation of these proteins in KRAS-dependent (KRAS-Dep) cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-Dep but not KRAS-independent NSCLCs. These results suggest that LCK is a potential druggable target protein in KRAS-Dep lung cancers. PMID:24135919

Effects of atorvastatin treatment on left ventricular diastolic function in peritoneal dialysis patients-The ALEVENT clinical trial.

PubMed

Wu, Cho-Kai; Yeh, Chih-Fan; Chiang, Jiun-Yang; Lin, Ting-Tse; Wu, Yi-Fan; Chiang, Chih-Kang; Kao, Tze-Wah; Hung, Kuan-Yu; Huang, Jenq-Wen

Left ventricular diastolic dysfunction (LVDD) is common among patients undergoing peritoneal dialysis (PD). Increased levels of inflammatory biomarkers, such as high-sensitivity C-reactive protein, predict the development of LVDD. We hypothesized that PD patients with elevated high-sensitivity C-reactive protein levels might benefit from statin treatment for LVDD and designed a randomized clinical trial to prove the hypothesis. We screened 213 PD patients and randomly assigned 32 men and women with low-density lipoprotein cholesterol levels <130 mg/dL, high-sensitivity C-reactive protein levels of ≥1.5 mg/L, and LVDD, diagnosed by conventional and tissue Doppler imaging (TDI) echocardiography, to treatment with atorvastatin, 40 mg daily, or without. The primary end points were changes in TDI diastolic parameters or global strain imaging diastolic parameters. Atorvastatin reduced low-density lipoprotein cholesterol levels by 43% and high-sensitivity C-reactive protein levels by 45% (both P < .001). Follow-up TDI showed significant improvement of early mitral flow velocities divided by early diastolic peak velocities of the mitral annulus at the medial and lateral site (Nominal change for E/E medial : -5.01 ± 6.36 vs 1.80 ± 6.59 for atorvastatin and control, respectively, P = .02). There was also a significant improvement in global strain imaging after atorvastatin treatment (global strain rate, -17.12 ± 1.42 vs -14.61 ± 1.78 for atorvastatin and control, respectively, P = .002 and E/SR IVR , 462.35 ± 110.54 vs 634.09 ± 116.81, P = .003). In this trial of PD patients without hyperlipidemia but with elevated high-sensitivity C-reactive protein levels and LVDD, atorvastatin significantly improved cardiac diastolic function (ClinicalTrials.gov number, NCT01503671). Copyright © 2017. Published by Elsevier Inc.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Soybean kinome: functional classification and gene expression patterns

PubMed Central

Liu, Jinyi; Chen, Nana; Grant, Joshua N.; Cheng, Zong-Ming (Max); Stewart, C. Neal; Hewezi, Tarek

2015-01-01

The protein kinase (PK) gene family is one of the largest and most highly conserved gene families in plants and plays a role in nearly all biological functions. While a large number of genes have been predicted to encode PKs in soybean, a comprehensive functional classification and global analysis of expression patterns of this large gene family is lacking. In this study, we identified the entire soybean PK repertoire or kinome, which comprised 2166 putative PK genes, representing 4.67% of all soybean protein-coding genes. The soybean kinome was classified into 19 groups, 81 families, and 122 subfamilies. The receptor-like kinase (RLK) group was remarkably large, containing 1418 genes. Collinearity analysis indicated that whole-genome segmental duplication events may have played a key role in the expansion of the soybean kinome, whereas tandem duplications might have contributed to the expansion of specific subfamilies. Gene structure, subcellular localization prediction, and gene expression patterns indicated extensive functional divergence of PK subfamilies. Global gene expression analysis of soybean PK subfamilies revealed tissue- and stress-specific expression patterns, implying regulatory functions over a wide range of developmental and physiological processes. In addition, tissue and stress co-expression network analysis uncovered specific subfamilies with narrow or wide interconnected relationships, indicative of their association with particular or broad signalling pathways, respectively. Taken together, our analyses provide a foundation for further functional studies to reveal the biological and molecular functions of PKs in soybean. PMID:25614662

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

A nitrogen response pathway regulates virulence functions in Fusarium oxysporum via the protein kinase TOR and the bZIP protein MeaB.

PubMed

López-Berges, Manuel S; Rispail, Nicolas; Prados-Rosales, Rafael C; Di Pietro, Antonio

2010-07-01

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source-independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the DeltameaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.

De novo pathogenic variants in CHAMP1 are associated with global developmental delay, intellectual disability, and dysmorphic facial features.

PubMed

Tanaka, Akemi J; Cho, Megan T; Retterer, Kyle; Jones, Julie R; Nowak, Catherine; Douglas, Jessica; Jiang, Yong-Hui; McConkie-Rosell, Allyn; Schaefer, G Bradley; Kaylor, Julie; Rahman, Omar A; Telegrafi, Aida; Friedman, Bethany; Douglas, Ganka; Monaghan, Kristin G; Chung, Wendy K

2016-01-01

We identified five unrelated individuals with significant global developmental delay and intellectual disability (ID), dysmorphic facial features and frequent microcephaly, and de novo predicted loss-of-function variants in chromosome alignment maintaining phosphoprotein 1 (CHAMP1). Our findings are consistent with recently reported de novo mutations in CHAMP1 in five other individuals with similar features. CHAMP1 is a zinc finger protein involved in kinetochore-microtubule attachment and is required for regulating the proper alignment of chromosomes during metaphase in mitosis. Mutations in CHAMP1 may affect cell division and hence brain development and function, resulting in developmental delay and ID.

Physical and molecular bases of protein thermal stability and cold adaptation.

PubMed

Pucci, Fabrizio; Rooman, Marianne

2017-02-01

The molecular bases of thermal and cold stability and adaptation, which allow proteins to remain folded and functional in the temperature ranges in which their host organisms live and grow, are still only partially elucidated. Indeed, both experimental and computational studies fail to yield a fully precise and global physical picture, essentially because all effects are context-dependent and thus quite intricate to unravel. We present a snapshot of the current state of knowledge of this highly complex and challenging issue, whose resolution would enable large-scale rational protein design. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression and in vitro functional analyses of recombinant Gam1 protein

PubMed Central

Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2014-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

Expression and in vitro functional analyses of recombinant Gam1 protein.

PubMed

Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2015-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Optimal network alignment with graphlet degree vectors.

PubMed

Milenković, Tijana; Ng, Weng Leong; Hayes, Wayne; Przulj, Natasa

2010-06-30

Important biological information is encoded in the topology of biological networks. Comparative analyses of biological networks are proving to be valuable, as they can lead to transfer of knowledge between species and give deeper insights into biological function, disease, and evolution. We introduce a new method that uses the Hungarian algorithm to produce optimal global alignment between two networks using any cost function. We design a cost function based solely on network topology and use it in our network alignment. Our method can be applied to any two networks, not just biological ones, since it is based only on network topology. We use our new method to align protein-protein interaction networks of two eukaryotic species and demonstrate that our alignment exposes large and topologically complex regions of network similarity. At the same time, our alignment is biologically valid, since many of the aligned protein pairs perform the same biological function. From the alignment, we predict function of yet unannotated proteins, many of which we validate in the literature. Also, we apply our method to find topological similarities between metabolic networks of different species and build phylogenetic trees based on our network alignment score. The phylogenetic trees obtained in this way bear a striking resemblance to the ones obtained by sequence alignments. Our method detects topologically similar regions in large networks that are statistically significant. It does this independent of protein sequence or any other information external to network topology.

Milk protein for improved metabolic health: a review of the evidence

PubMed Central

2013-01-01

Epidemiological evidence shows that consumption of dairy products is associated with decreased prevalence of metabolic related disorders, whilst evidence from experimental studies points towards dairy protein as a dietary component which may aid prevention of type 2 diabetes (T2DM). Poor metabolic health is a common characteristic of overweight, obesity and aging, and is the forerunner of T2DM and cardiovascular disease (CVD), and an ever increasing global health issue. Progressive loss of metabolic control is evident from a blunting of carbohydrate, fat and protein metabolism, which is commonly manifested through decreased insulin sensitivity, inadequate glucose and lipid control, accompanied by a pro-inflammatory environment and hypertension. Adverse physiological changes such as excess visceral adipose tissue deposition and expansion, lipid overspill and infiltration into liver, muscle and other organs, and sarcopaenia or degenerative loss of skeletal muscle mass and function all underpin this adverse profile. ‘Sarcobesity’ and sarcopaenic diabetes are rapidly growing health issues. As well as through direct mechanisms, dairy protein may indirectly improve metabolic health by aiding loss of body weight and fat mass through enhanced satiety, whilst promoting skeletal muscle growth and function through anabolic effects of dairy protein-derived branch chain amino acids (BCAAs). BCAAs enhance muscle protein synthesis, lean body mass and skeletal muscle metabolic function. The composition and processing of dairy protein has an impact on digestion, absorption, BCAA kinetics and function, hence the optimisation of dairy protein composition through selection and combination of specific protein components in milk may provide a way to maximize benefits for metabolic health. PMID:23822206

Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

PubMed

Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

2009-02-01

The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

Functional Comparison of Bacteria from the Human Gut and Closely Related Non-Gut Bacteria Reveals the Importance of Conjugation and a Paucity of Motility and Chemotaxis Functions in the Gut Environment.

PubMed

Dobrijevic, Dragana; Abraham, Anne-Laure; Jamet, Alexandre; Maguin, Emmanuelle; van de Guchte, Maarten

2016-01-01

The human GI tract is a complex and still poorly understood environment, inhabited by one of the densest microbial communities on earth. The gut microbiota is shaped by millennia of evolution to co-exist with the host in commensal or symbiotic relationships. Members of the gut microbiota perform specific molecular functions important in the human gut environment. This can be illustrated by the presence of a highly expanded repertoire of proteins involved in carbohydrate metabolism, in phase with the large diversity of polysaccharides originating from the diet or from the host itself that can be encountered in this environment. In order to identify other bacterial functions that are important in the human gut environment, we investigated the distribution of functional groups of proteins in a group of human gut bacteria and their close non-gut relatives. Complementary to earlier global comparisons between different ecosystems, this approach should allow a closer focus on a group of functions directly related to the gut environment while avoiding functions related to taxonomically divergent microbiota composition, which may or may not be relevant for gut homeostasis. We identified several functions that are overrepresented in the human gut bacteria which had not been recognized in a global approach. The observed under-representation of certain other functions may be equally important for gut homeostasis. Together, these analyses provide us with new information about this environment so critical to our health and well-being.

Functional Comparison of Bacteria from the Human Gut and Closely Related Non-Gut Bacteria Reveals the Importance of Conjugation and a Paucity of Motility and Chemotaxis Functions in the Gut Environment

PubMed Central

Dobrijevic, Dragana; Abraham, Anne-Laure; Jamet, Alexandre; Maguin, Emmanuelle; van de Guchte, Maarten

2016-01-01

The human GI tract is a complex and still poorly understood environment, inhabited by one of the densest microbial communities on earth. The gut microbiota is shaped by millennia of evolution to co-exist with the host in commensal or symbiotic relationships. Members of the gut microbiota perform specific molecular functions important in the human gut environment. This can be illustrated by the presence of a highly expanded repertoire of proteins involved in carbohydrate metabolism, in phase with the large diversity of polysaccharides originating from the diet or from the host itself that can be encountered in this environment. In order to identify other bacterial functions that are important in the human gut environment, we investigated the distribution of functional groups of proteins in a group of human gut bacteria and their close non-gut relatives. Complementary to earlier global comparisons between different ecosystems, this approach should allow a closer focus on a group of functions directly related to the gut environment while avoiding functions related to taxonomically divergent microbiota composition, which may or may not be relevant for gut homeostasis. We identified several functions that are overrepresented in the human gut bacteria which had not been recognized in a global approach. The observed under-representation of certain other functions may be equally important for gut homeostasis. Together, these analyses provide us with new information about this environment so critical to our health and well-being. PMID:27416027

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

PubMed

Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

2016-02-01

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

[Chlamydia trachomatis proteasome protein as one of the significant pathogenicity factors of exciter].

PubMed

Davydov, D Iu; Zigangirova, N A

2014-01-01

Sex-related infections are a global problem. Such infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. Protein CPAF is leading factor of pathogenesis. This protein inhibits the signaling pathways of host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review general properties of the proteasome Chlamydia protein CPAF, its functions, and role in pathology. The role of protein CPAF in the anti-chlamydia immune reaction is discussed. The prospects of the development of promising anti-chlamydia vaccine, as well as new effective anti-chlamydia drugs are also discussed.

Mutual information estimation reveals global associations between stimuli and biological processes

PubMed Central

Suzuki, Taiji; Sugiyama, Masashi; Kanamori, Takafumi; Sese, Jun

2009-01-01

Background Although microarray gene expression analysis has become popular, it remains difficult to interpret the biological changes caused by stimuli or variation of conditions. Clustering of genes and associating each group with biological functions are often used methods. However, such methods only detect partial changes within cell processes. Herein, we propose a method for discovering global changes within a cell by associating observed conditions of gene expression with gene functions. Results To elucidate the association, we introduce a novel feature selection method called Least-Squares Mutual Information (LSMI), which computes mutual information without density estimaion, and therefore LSMI can detect nonlinear associations within a cell. We demonstrate the effectiveness of LSMI through comparison with existing methods. The results of the application to yeast microarray datasets reveal that non-natural stimuli affect various biological processes, whereas others are no significant relation to specific cell processes. Furthermore, we discover that biological processes can be categorized into four types according to the responses of various stimuli: DNA/RNA metabolism, gene expression, protein metabolism, and protein localization. Conclusion We proposed a novel feature selection method called LSMI, and applied LSMI to mining the association between conditions of yeast and biological processes through microarray datasets. In fact, LSMI allows us to elucidate the global organization of cellular process control. PMID:19208155

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

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

Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

PubMed

Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

2014-01-01

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

The Potential for Engineering Enhanced Functional-Feed Soybeans for Sustainable Aquaculture Feed.

PubMed

Herman, Eliot M; Schmidt, Monica A

2016-01-01

Aquaculture is the most rapidly growing segment of global animal production that now surpasses wild-capture fisheries production and is continuing to grow 10% annually. Sustainable aquaculture needs to diminish, and progressively eliminate, its dependence on fishmeal-sourced feed from over-harvested fisheries. Sustainable aquafeed sources will need to be primarily of plant-origin. Soybean is currently the primary global vegetable-origin protein source for aquaculture. Direct exchange of soybean meal for fishmeal in aquafeed has resulted in reduced growth rates due in part to soybean's anti-nutritional proteins. To produce soybeans for use in aquaculture feeds a new conventional line has been bred termed Triple Null by stacking null alleles for the feed-relevant proteins Kunitz Trypsin Inhibitor, lectin, and P34 allergen. Triple Null is now being further enhanced as a platform to build additional transgene traits for vaccines, altered protein composition, and to produce high levels of β-carotene an intrinsic orange-colored aquafeed marker to distinguish the seeds from commodity beans and as the metabolic feedstock precursor of highly valued astaxanthin.

Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

NASA Astrophysics Data System (ADS)

McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

2015-03-01

We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report

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

Summers, Anne O.; Miller, Susan M.; Wall, Judy

2016-06-18

Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we have devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolicmore » functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation). We have also discovered that acute Hg exposure severely disrupts thiol, iron and redox homeostases, and electrolyte balance (LaVoie, et al., 2015) Thus, we proposed to bring these techniques to bear on the central problem of identifying the cellular proteins involved in bacterial uptake and methylation of mercury and its release from the cell.« less

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

Population reversal driven by unrestrained interactions in molecular dynamics simulations: A dialanine model

NASA Astrophysics Data System (ADS)

Pullara, Filippo; Ignacio, J., General

2015-10-01

Standard Molecular Dynamics simulations (MD) are usually performed under periodic boundary conditions using the well-established "Ewald summation". This implies that the distance among each element in a given lattice cell and its corresponding element in another cell, as well as their relative orientations, are constant. Consequently, protein-protein interactions between proteins in different cells—important in many biological activities, such as protein cooperativity and physiological/pathological aggregation—are severely restricted, and features driven by protein-protein interactions are lost. The consequences of these restrictions, although conceptually understood and mentioned in the literature, have not been quantitatively studied before. The effect of protein-protein interactions on the free energy landscape of a model system, dialanine, is presented. This simple system features a free energy diagram with well-separated minima. It is found that, in the case of absence of peptide-peptide (p-p) interactions, the ψ = 150° dihedral angle determines the most energetically favored conformation (global free-energy minimum). When strong p-p interactions are induced, the global minimum switches to the ψ = 0° conformation. This shows that the free-energy landscape of an individual molecule is dramatically affected by the presence of other freely interacting molecules of its same type. Results of the study suggest how taking into account p-p interactions in MD allows having a more realistic picture of system activity and functional conformations.

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)*

PubMed Central

Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A.; Schoenhard, Grant; Zemelman, Boris V.; Mechref, Yehia; Raab-Graham, Kimberly F.

2016-01-01

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. PMID:26419955

FACETS: multi-faceted functional decomposition of protein interaction networks.

PubMed

Seah, Boon-Siew; Bhowmick, Sourav S; Dewey, C Forbes

2012-10-15

The availability of large-scale curated protein interaction datasets has given rise to the opportunity to investigate higher level organization and modularity within the protein-protein interaction (PPI) network using graph theoretic analysis. Despite the recent progress, systems level analysis of high-throughput PPIs remains a daunting task because of the amount of data they present. In this article, we propose a novel PPI network decomposition algorithm called FACETS in order to make sense of the deluge of interaction data using Gene Ontology (GO) annotations. FACETS finds not just a single functional decomposition of the PPI network, but a multi-faceted atlas of functional decompositions that portray alternative perspectives of the functional landscape of the underlying PPI network. Each facet in the atlas represents a distinct interpretation of how the network can be functionally decomposed and organized. Our algorithm maximizes interpretative value of the atlas by optimizing inter-facet orthogonality and intra-facet cluster modularity. We tested our algorithm on the global networks from IntAct, and compared it with gold standard datasets from MIPS and KEGG. We demonstrated the performance of FACETS. We also performed a case study that illustrates the utility of our approach. Supplementary data are available at the Bioinformatics online. Our software is available freely for non-commercial purposes from: http://www.cais.ntu.edu.sg/~assourav/Facets/

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-03-19

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Simulating protein folding initiation sites using an alpha-carbon-only knowledge-based force field

PubMed Central

Buck, Patrick M.; Bystroff, Christopher

2015-01-01

Protein folding is a hierarchical process where structure forms locally first, then globally. Some short sequence segments initiate folding through strong structural preferences that are independent of their three-dimensional context in proteins. We have constructed a knowledge-based force field in which the energy functions are conditional on local sequence patterns, as expressed in the hidden Markov model for local structure (HMMSTR). Carbon-alpha force field (CALF) builds sequence specific statistical potentials based on database frequencies for α-carbon virtual bond opening and dihedral angles, pairwise contacts and hydrogen bond donor-acceptor pairs, and simulates folding via Brownian dynamics. We introduce hydrogen bond donor and acceptor potentials as α-carbon probability fields that are conditional on the predicted local sequence. Constant temperature simulations were carried out using 27 peptides selected as putative folding initiation sites, each 12 residues in length, representing several different local structure motifs. Each 0.6 μs trajectory was clustered based on structure. Simulation convergence or representativeness was assessed by subdividing trajectories and comparing clusters. For 21 of the 27 sequences, the largest cluster made up more than half of the total trajectory. Of these 21 sequences, 14 had cluster centers that were at most 2.6 Å root mean square deviation (RMSD) from their native structure in the corresponding full-length protein. To assess the adequacy of the energy function on nonlocal interactions, 11 full length native structures were relaxed using Brownian dynamics simulations. Equilibrated structures deviated from their native states but retained their overall topology and compactness. A simple potential that folds proteins locally and stabilizes proteins globally may enable a more realistic understanding of hierarchical folding pathways. PMID:19137613

Integrated proteomics and metabolomics analysis of rat testis: Mechanism of arsenic-induced male reproductive toxicity

NASA Astrophysics Data System (ADS)

Huang, Qingyu; Luo, Lianzhong; Alamdar, Ambreen; Zhang, Jie; Liu, Liangpo; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

2016-09-01

Arsenic is a widespread metalloid in environment, whose exposure has been associated with a broad spectrum of toxic effects. However, a global view of arsenic-induced male reproductive toxicity is still lack, and the underlying mechanisms remain largely unclear. Our results revealed that arsenic exposure decreased testosterone level and reduced sperm quality in rats. By conducting an integrated proteomics and metabolomics analysis, the present study aims to investigate the global influence of arsenic exposure on the proteome and metabolome in rat testis. The abundance of 70 proteins (36 up-regulated and 34 down-regulated) and 13 metabolites (8 increased and 5 decreased) were found to be significantly altered by arsenic treatment. Among these, 19 proteins and 2 metabolites were specifically related to male reproductive system development and function, including spermatogenesis, sperm function and fertilization, fertility, internal genitalia development, and mating behavior. It is further proposed that arsenic mainly impaired spermatogenesis and fertilization via aberrant modulation of these male reproduction-related proteins and metabolites, which may be mediated by the ERK/AKT/NF-κB-dependent signaling pathway. Overall, these findings will aid our understanding of the mechanisms responsible for arsenic-induced male reproductive toxicity, and from such studies useful biomarkers indicative of arsenic exposure could be discovered.

Integrated proteomics and metabolomics analysis of rat testis: Mechanism of arsenic-induced male reproductive toxicity.

PubMed

Huang, Qingyu; Luo, Lianzhong; Alamdar, Ambreen; Zhang, Jie; Liu, Liangpo; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

2016-09-02

Arsenic is a widespread metalloid in environment, whose exposure has been associated with a broad spectrum of toxic effects. However, a global view of arsenic-induced male reproductive toxicity is still lack, and the underlying mechanisms remain largely unclear. Our results revealed that arsenic exposure decreased testosterone level and reduced sperm quality in rats. By conducting an integrated proteomics and metabolomics analysis, the present study aims to investigate the global influence of arsenic exposure on the proteome and metabolome in rat testis. The abundance of 70 proteins (36 up-regulated and 34 down-regulated) and 13 metabolites (8 increased and 5 decreased) were found to be significantly altered by arsenic treatment. Among these, 19 proteins and 2 metabolites were specifically related to male reproductive system development and function, including spermatogenesis, sperm function and fertilization, fertility, internal genitalia development, and mating behavior. It is further proposed that arsenic mainly impaired spermatogenesis and fertilization via aberrant modulation of these male reproduction-related proteins and metabolites, which may be mediated by the ERK/AKT/NF-κB-dependent signaling pathway. Overall, these findings will aid our understanding of the mechanisms responsible for arsenic-induced male reproductive toxicity, and from such studies useful biomarkers indicative of arsenic exposure could be discovered.

Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks

PubMed Central

2012-01-01

Background Age-related macular degeneration (AMD) is a leading cause of blindness that affects the central region of the retinal pigmented epithelium (RPE), choroid, and neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD leads to progressive retinal degeneration, and in advanced cases, irreversible vision loss. Although genetic analysis, animal models, and cell culture systems have yielded important insights into AMD, the molecular pathways underlying AMD's onset and progression remain poorly delineated. We sought to better understand the molecular underpinnings of this devastating disease by performing the first comparative transcriptome analysis of AMD and normal human donor eyes. Methods RPE-choroid and retina tissue samples were obtained from a common cohort of 31 normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles were generated for macular and extramacular regions, and statistical and bioinformatic methods were employed to identify disease-associated gene signatures and functionally enriched protein association networks. Selected genes of high significance were validated using an independent donor cohort. Results We identified over 50 annotated genes enriched in cell-mediated immune responses that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine learning model and a second donor cohort, we show that the top 20 global genes are predictive of AMD clinical diagnosis. We also discovered functionally enriched gene sets in the RPE-choroid that delineate the advanced AMD phenotypes, neovascular AMD and geographic atrophy. Moreover, we identified a graded increase of transcript levels in the retina related to wound response, complement cascade, and neurogenesis that strongly correlates with decreased levels of phototransduction transcripts and increased AMD severity. Based on our findings, we assembled protein-protein interactomes that highlight functional networks likely to be involved in AMD pathogenesis. Conclusions We discovered new global biomarkers and gene expression signatures of AMD. These results are consistent with a model whereby cell-based inflammatory responses represent a central feature of AMD etiology, and depending on genetics, environment, or stochastic factors, may give rise to the advanced AMD phenotypes characterized by angiogenesis and/or cell death. Genes regulating these immunological activities, along with numerous other genes identified here, represent promising new targets for AMD-directed therapeutics and diagnostics. Please see related commentary: http://www.biomedcentral.com/1741-7015/10/21/abstract PMID:22364233

Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism.

PubMed

Matsuda, Fumio; Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

2017-01-01

Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.

Chronic Cigarette Smoke Mediated Global Changes in Lung Mucoepidermoid Cells: A Phosphoproteomic Analysis.

PubMed

Solanki, Hitendra S; Advani, Jayshree; Khan, Aafaque Ahmad; Radhakrishnan, Aneesha; Sahasrabuddhe, Nandini A; Pinto, Sneha M; Chang, Xiaofei; Prasad, Thottethodi Subrahmanya Keshava; Mathur, Premendu Prakash; Sidransky, David; Gowda, Harsha; Chatterjee, Aditi

2017-08-01

Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers.

Global analysis of bacterial transcription factors to predict cellular target processes.

PubMed

Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

2004-03-01

Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs.

A computational interactome for prioritizing genes associated with complex agronomic traits in rice (Oryza sativa).

PubMed

Liu, Shiwei; Liu, Yihui; Zhao, Jiawei; Cai, Shitao; Qian, Hongmei; Zuo, Kaijing; Zhao, Lingxia; Zhang, Lida

2017-04-01

Rice (Oryza sativa) is one of the most important staple foods for more than half of the global population. Many rice traits are quantitative, complex and controlled by multiple interacting genes. Thus, a full understanding of genetic relationships will be critical to systematically identify genes controlling agronomic traits. We developed a genome-wide rice protein-protein interaction network (RicePPINet, http://netbio.sjtu.edu.cn/riceppinet) using machine learning with structural relationship and functional information. RicePPINet contained 708 819 predicted interactions for 16 895 non-transposable element related proteins. The power of the network for discovering novel protein interactions was demonstrated through comparison with other publicly available protein-protein interaction (PPI) prediction methods, and by experimentally determined PPI data sets. Furthermore, global analysis of domain-mediated interactions revealed RicePPINet accurately reflects PPIs at the domain level. Our studies showed the efficiency of the RicePPINet-based method in prioritizing candidate genes involved in complex agronomic traits, such as disease resistance and drought tolerance, was approximately 2-11 times better than random prediction. RicePPINet provides an expanded landscape of computational interactome for the genetic dissection of agronomically important traits in rice. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Local and global influences on protein turnover in neurons and glia

PubMed Central

Dörrbaum, Aline R; Kochen, Lisa

2018-01-01

Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover. PMID:29914620

Charting organellar importomes by quantitative mass spectrometry

PubMed Central

Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina

2017-01-01

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles. PMID:28485388

Impact of Pyrrolidine Dithiocarbamate and Interleukin-6 on Mammalian Target of Rapamycin Complex 1 Regulation and Global Protein TranslationS⃞

PubMed Central

Song, Shaoming; Abdelmohsen, Kotb; Zhang, Yongqing; Becker, Kevin G.; Gorospe, Myriam

2011-01-01

Interleukin-6 (IL-6) is a proinflammatory cytokine that exerts a wide range of cellular, physiological, and pathophysiological responses. Pyrrolidine dithiocarbamate (PDTC) antagonizes the cellular responsiveness to IL-6 through impairment in signal transducer and activator of transcription-3 activation and downstream signaling. To further elucidate the biological properties of PDTC, global gene expression profiling of human HepG2 hepatocellular carcinoma cells was carried out after treatment with PDTC or IL-6 for up to 8 h. Through an unbiased pathway analysis method, gene array analysis showed dramatic and temporal differences in expression changes in response to PDTC versus IL-6. A significant number of genes associated with metabolic pathways, inflammation, translation, and mitochondrial function were changed, with ribosomal protein genes and DNA damage-inducible transcript 4 protein (DDIT4) primarily up-regulated with PDTC but down-regulated with IL-6. Quantitative polymerase chain reaction and Western blot analyses validated the microarray data and showed the reciprocal expression pattern of the mammalian target of rapamycin (mTOR)-negative regulator DDIT4 in response to PDTC versus IL-6. Cell treatment with PDTC resulted in a rapid and sustained activation of Akt and subsequently blocked the IL-6-mediated increase in mTOR complex 1 function through up-regulation in DDIT4 expression. Conversely, down-regulation of DDIT4 with small interfering RNA dampened the capacity of PDTC to block IL-6-dependent mTOR activation. The overall protein biosynthetic capacity of the cells was severely blunted by IL-6 but increased in a rapamycin-independent pathway by PDTC. These results demonstrate a critical effect of PDTC on mTOR complex 1 function and provide evidence that PDTC can reverse IL-6-related signaling via induction of DDIT4. PMID:21917559

Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

PubMed Central

Wang, Huaishan; Yang, Jia; Yang, Qi; Fu, Yi; Hu, Yu; Liu, Fang; Wang, Weiqing; Cui, Lianxian; Chen, Hui; Zhang, Jianmin; He, Wei

2016-01-01

Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis. PMID:27907096

Actin–microtubule coordination at growing microtubule ends

PubMed Central

López, Magdalena Preciado; Huber, Florian; Grigoriev, Ilya; Steinmetz, Michel O.; Akhmanova, Anna; Koenderink, Gijsje H.; Dogterom, Marileen

2014-01-01

To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts. PMID:25159196

Fibrillation mechanism of a model intrinsically disordered protein revealed by 2D correlation deep UV resonance Raman spectroscopy.

PubMed

Sikirzhytski, Vitali; Topilina, Natalya I; Takor, Gaius A; Higashiya, Seiichiro; Welch, John T; Uversky, Vladimir N; Lednev, Igor K

2012-05-14

Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of β-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and β-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.

Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia

PubMed Central

Ibebunjo, Chikwendu; Chick, Joel M.; Kendall, Tracee; Eash, John K.; Li, Christine; Zhang, Yunyu; Vickers, Chad; Wu, Zhidan; Clarke, Brian A.; Shi, Jun; Cruz, Joseph; Fournier, Brigitte; Brachat, Sophie; Gutzwiller, Sabine; Ma, QiCheng; Markovits, Judit; Broome, Michelle; Steinkrauss, Michelle; Skuba, Elizabeth; Galarneau, Jean-Rene; Gygi, Steven P.

2013-01-01

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia. PMID:23109432

An integrated proteomic approach uncovers novel substrates and functions of the Lon protease in Escherichia coli.

PubMed

Arends, Jan; Griego, Marcena; Thomanek, Nikolas; Lindemann, Claudia; Kutscher, Blanka; Meyer, Helmut E; Narberhaus, Franz

2018-04-30

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example the superoxide-stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

PubMed Central

Engen, John R.; Wales, Thomas E.; Chen, Shugui; Marzluff, Elaine M.; Hassell, Kerry M.; Weis, David D.; Smithgall, Thomas E.

2013-01-01

Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion. PMID:23682200

Redox responses are preserved across muscle fibres with differential susceptibility to aging.

PubMed

Smith, Neil T; Soriano-Arroquia, Ana; Goljanek-Whysall, Katarzyna; Jackson, Malcolm J; McDonagh, Brian

2018-04-15

Age-related loss of muscle mass and function is associated with increased frailty and loss of independence. The mechanisms underlying the susceptibility of different muscle types to age-related atrophy are not fully understood. Reactive oxygen species (ROS) are recognised as important signalling molecules in healthy muscle and redox sensitive proteins can respond to intracellular changes in ROS concentrations modifying reactive thiol groups on Cysteine (Cys) residues. Conserved Cys residues tend to occur in functionally important locations and can have a direct impact on protein function through modifications at the active site or determining protein conformation. The aim of this work was to determine age-related changes in the redox proteome of two metabolically distinct murine skeletal muscles, the quadriceps a predominantly glycolytic muscle and the soleus which contains a higher proportion of mitochondria. To examine the effects of aging on the global proteome and the oxidation state of individual redox sensitive Cys residues, we employed a label free proteomics approach including a differential labelling of reduced and reversibly oxidised Cys residues. Our results indicate the proteomic response to aging is dependent on muscle type but redox changes that occur primarily in metabolic and cytoskeletal proteins are generally preserved between metabolically distinct tissues. Skeletal muscle containing fast twitch glycolytic fibres are more susceptible to age related atrophy compared to muscles with higher proportions of oxidative slow twitch fibres. Contracting skeletal muscle generates reactive oxygen species that are required for correct signalling and adaptation to exercise and it is also known that the intracellular redox environment changes with age. To identify potential mechanisms for the distinct response to age, this article combines a global proteomic approach and a differential labelling of reduced and reversibly oxidised Cysteine residues in two metabolically distinct skeletal muscles, quadriceps and soleus, from adult and old mice. Our results indicate that the global proteomic changes with age in skeletal muscles are dependent on fibre type. However, redox specific changes are preserved across muscle types and accompanied with a reduction in the number of redox sensitive Cysteine residues. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Towards quantitative classification of folded proteins in terms of elementary functions.

PubMed

Hu, Shuangwei; Krokhotin, Andrei; Niemi, Antti J; Peng, Xubiao

2011-04-01

A comparative classification scheme provides a good basis for several approaches to understand proteins, including prediction of relations between their structure and biological function. But it remains a challenge to combine a classification scheme that describes a protein starting from its well-organized secondary structures and often involves direct human involvement, with an atomary-level physics-based approach where a protein is fundamentally nothing more than an ensemble of mutually interacting carbon, hydrogen, oxygen, and nitrogen atoms. In order to bridge these two complementary approaches to proteins, conceptually novel tools need to be introduced. Here we explain how an approach toward geometric characterization of entire folded proteins can be based on a single explicit elementary function that is familiar from nonlinear physical systems where it is known as the kink soliton. Our approach enables the conversion of hierarchical structural information into a quantitative form that allows for a folded protein to be characterized in terms of a small number of global parameters that are in principle computable from atomary-level considerations. As an example we describe in detail how the native fold of the myoglobin 1M6C emerges from a combination of kink solitons with a very high atomary-level accuracy. We also verify that our approach describes longer loops and loops connecting α helices with β strands, with the same overall accuracy. ©2011 American Physical Society

Gαi Proteins are Indispensable for Hearing.

PubMed

Beer-Hammer, Sandra; Lee, Sze Chim; Mauriac, Stephanie A; Leiss, Veronika; Groh, Isabel A M; Novakovic, Ana; Piekorz, Roland P; Bucher, Kirsten; Chen, Chengfang; Ni, Kun; Singer, Wibke; Harasztosi, Csaba; Schimmang, Thomas; Zimmermann, Ulrike; Pfeffer, Klaus; Birnbaumer, Lutz; Forge, Andrew; Montcouquiol, Mireille; Knipper, Marlies; Nürnberg, Bernd; Rüttiger, Lukas

2018-06-21

From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes. © 2018 The Author(s). Published by S. Karger AG, Basel.

Mapping hydration dynamics and coupled water-protein fluctuations around a protein surface

NASA Astrophysics Data System (ADS)

Zhang, Luyuan; Wang, Lijuan; Kao, Ya-Ting; Qiu, Weihong; Yang, Yi; Okobiah, Oghaghare; Zhong, Dongping

2009-03-01

Elucidation of the molecular mechanism of water-protein interactions is critical to understanding many fundamental aspects of protein science, such as protein folding and misfolding and enzyme catalysis. We recently carried out a global mapping of protein-surface hydration dynamics around a globular α-helical protein apomyoglobin. The intrinsic optical probe tryptophan was employed to scan the protein surface one at a time by site-specific mutagenesis. With femtosecond resolution, we mapped out the dynamics of water-protein interactions with more than 20 mutants and for two states, native and molten globular. A robust bimodal distribution of time scales was observed, representing two types of water motions: local relaxation and protein-coupled fluctuations. The time scales show a strong correlation with the local protein structural rigidity and chemical identity. We also resolved two distinct contributions to the overall Stokes-shifts from the two time scales. These results are significant to understanding the role of hydration water on protein structural stability, dynamics and function.

Mixture models for protein structure ensembles.

PubMed

Hirsch, Michael; Habeck, Michael

2008-10-01

Protein structure ensembles provide important insight into the dynamics and function of a protein and contain information that is not captured with a single static structure. However, it is not clear a priori to what extent the variability within an ensemble is caused by internal structural changes. Additional variability results from overall translations and rotations of the molecule. And most experimental data do not provide information to relate the structures to a common reference frame. To report meaningful values of intrinsic dynamics, structural precision, conformational entropy, etc., it is therefore important to disentangle local from global conformational heterogeneity. We consider the task of disentangling local from global heterogeneity as an inference problem. We use probabilistic methods to infer from the protein ensemble missing information on reference frames and stable conformational sub-states. To this end, we model a protein ensemble as a mixture of Gaussian probability distributions of either entire conformations or structural segments. We learn these models from a protein ensemble using the expectation-maximization algorithm. Our first model can be used to find multiple conformers in a structure ensemble. The second model partitions the protein chain into locally stable structural segments or core elements and less structured regions typically found in loops. Both models are simple to implement and contain only a single free parameter: the number of conformers or structural segments. Our models can be used to analyse experimental ensembles, molecular dynamics trajectories and conformational change in proteins. The Python source code for protein ensemble analysis is available from the authors upon request.

Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings.

PubMed

Engelsberger, Wolfgang R; Schulze, Waltraud X

2012-03-01

Nitrogen is an essential macronutrient for plant growth and development. Inorganic nitrogen and its assimilation products control various metabolic, physiological and developmental processes. Although the transcriptional responses induced by nitrogen have been extensively studied in the past, our work here focused on the discovery of candidate proteins for regulatory events that are complementary to transcriptional changes. Most signaling pathways involve modulation of protein abundance and/or activity by protein phosphorylation. Therefore, we analyzed the dynamic changes in protein phosphorylation in membrane and soluble proteins from plants exposed to rapid changes in nutrient availability over a time course of 30 min. Plants were starved of nitrogen and subsequently resupplied with nitrogen in the form of nitrate or ammonium. Proteins with maximum change in their phosphorylation level at up to 5 min after nitrogen resupply (fast responses) included GPI-anchored proteins, receptor kinases and transcription factors, while proteins with maximum change in their phosphorylation level after 10 min of nitrogen resupply (late responses) included proteins involved in protein synthesis and degradation, as well as proteins with functions in central metabolism and hormone metabolism. Resupply of nitrogen in the form of nitrate or ammonium resulted in distinct phosphorylation patterns, mainly of proteins with signaling functions, transcription factors and transporters. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Heat Shock Proteins in Association with Heat Tolerance in Grasses

PubMed Central

Xu, Yan; Zhan, Chenyang; Huang, Bingru

2011-01-01

The grass family Poaceae includes annual species cultivated as major grain crops and perennial species cultivated as forage or turf grasses. Heat stress is a primary factor limiting growth and productivity of cool-season grass species and is becoming a more significant problem in the context of global warming. Plants have developed various mechanisms in heat-stress adaptation, including changes in protein metabolism such as the induction of heat shock proteins (HSPs). This paper summarizes the structure and function of major HSPs, recent research progress on the association of HSPs with grass tolerance to heat stress, and incorporation of HSPs in heat-tolerant grass breeding. PMID:22084689

A large dataset of protein dynamics in the mammalian heart proteome.

PubMed

Lau, Edward; Cao, Quan; Ng, Dominic C M; Bleakley, Brian J; Dincer, T Umut; Bot, Brian M; Wang, Ding; Liem, David A; Lam, Maggie P Y; Ge, Junbo; Ping, Peipei

2016-03-15

Protein stability is a major regulatory principle of protein function and cellular homeostasis. Despite limited understanding on mechanisms, disruption of protein turnover is widely implicated in diverse pathologies from heart failure to neurodegenerations. Information on global protein dynamics therefore has the potential to expand the depth and scope of disease phenotyping and therapeutic strategies. Using an integrated platform of metabolic labeling, high-resolution mass spectrometry and computational analysis, we report here a comprehensive dataset of the in vivo half-life of 3,228 and the expression of 8,064 cardiac proteins, quantified under healthy and hypertrophic conditions across six mouse genetic strains commonly employed in biomedical research. We anticipate these data will aid in understanding key mitochondrial and metabolic pathways in heart diseases, and further serve as a reference for methodology development in dynamics studies in multiple organ systems.

A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB[C][W

PubMed Central

López-Berges, Manuel S.; Rispail, Nicolas; Prados-Rosales, Rafael C.; Di Pietro, Antonio

2010-01-01

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source–independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the ΔmeaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi. PMID:20639450

Protein loop modeling using a new hybrid energy function and its application to modeling in inaccurate structural environments.

PubMed

Park, Hahnbeom; Lee, Gyu Rie; Heo, Lim; Seok, Chaok

2014-01-01

Protein loop modeling is a tool for predicting protein local structures of particular interest, providing opportunities for applications involving protein structure prediction and de novo protein design. Until recently, the majority of loop modeling methods have been developed and tested by reconstructing loops in frameworks of experimentally resolved structures. In many practical applications, however, the protein loops to be modeled are located in inaccurate structural environments. These include loops in model structures, low-resolution experimental structures, or experimental structures of different functional forms. Accordingly, discrepancies in the accuracy of the structural environment assumed in development of the method and that in practical applications present additional challenges to modern loop modeling methods. This study demonstrates a new strategy for employing a hybrid energy function combining physics-based and knowledge-based components to help tackle this challenge. The hybrid energy function is designed to combine the strengths of each energy component, simultaneously maintaining accurate loop structure prediction in a high-resolution framework structure and tolerating minor environmental errors in low-resolution structures. A loop modeling method based on global optimization of this new energy function is tested on loop targets situated in different levels of environmental errors, ranging from experimental structures to structures perturbed in backbone as well as side chains and template-based model structures. The new method performs comparably to force field-based approaches in loop reconstruction in crystal structures and better in loop prediction in inaccurate framework structures. This result suggests that higher-accuracy predictions would be possible for a broader range of applications. The web server for this method is available at http://galaxy.seoklab.org/loop with the PS2 option for the scoring function.

Structural symmetry and protein function.

PubMed

Goodsell, D S; Olson, A J

2000-01-01

The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of any symmetry.

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

PubMed Central

Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup

2015-01-01

ABSTRACT Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:25995028

Normal mode-guided transition pathway generation in proteins

PubMed Central

Lee, Byung Ho; Seo, Sangjae; Kim, Min Hyeok; Kim, Youngjin; Jo, Soojin; Choi, Moon-ki; Lee, Hoomin; Choi, Jae Boong

2017-01-01

The biological function of proteins is closely related to its structural motion. For instance, structurally misfolded proteins do not function properly. Although we are able to experimentally obtain structural information on proteins, it is still challenging to capture their dynamics, such as transition processes. Therefore, we need a simulation method to predict the transition pathways of a protein in order to understand and study large functional deformations. Here, we present a new simulation method called normal mode-guided elastic network interpolation (NGENI) that performs normal modes analysis iteratively to predict transition pathways of proteins. To be more specific, NGENI obtains displacement vectors that determine intermediate structures by interpolating the distance between two end-point conformations, similar to a morphing method called elastic network interpolation. However, the displacement vector is regarded as a linear combination of the normal mode vectors of each intermediate structure, in order to enhance the physical sense of the proposed pathways. As a result, we can generate more reasonable transition pathways geometrically and thermodynamically. By using not only all normal modes, but also in part using only the lowest normal modes, NGENI can still generate reasonable pathways for large deformations in proteins. This study shows that global protein transitions are dominated by collective motion, which means that a few lowest normal modes play an important role in this process. NGENI has considerable merit in terms of computational cost because it is possible to generate transition pathways by partial degrees of freedom, while conventional methods are not capable of this. PMID:29020017

Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.

PubMed

Lee, Kenneth K; Sardiu, Mihaela E; Swanson, Selene K; Gilmore, Joshua M; Torok, Michael; Grant, Patrick A; Florens, Laurence; Workman, Jerry L; Washburn, Michael P

2011-07-05

Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.

Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes

PubMed Central

Lee, Kenneth K; Sardiu, Mihaela E; Swanson, Selene K; Gilmore, Joshua M; Torok, Michael; Grant, Patrick A; Florens, Laurence; Workman, Jerry L; Washburn, Michael P

2011-01-01

Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt–Ada–Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants. PMID:21734642

Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications.

PubMed

Jimenez, Connie R; Verheul, Henk M W

2014-01-01

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

Prediction of protein tertiary structure to low resolution: performance for a large and structurally diverse test set.

PubMed

Eyrich, V A; Standley, D M; Friesner, R A

1999-05-14

We report the tertiary structure predictions for 95 proteins ranging in size from 17 to 160 residues starting from known secondary structure. Predictions are obtained from global minimization of an empirical potential function followed by the application of a refined atomic overlap potential. The minimization strategy employed represents a variant of the Monte Carlo plus minimization scheme of Li and Scheraga applied to a reduced model of the protein chain. For all of the cases except beta-proteins larger than 75 residues, a native-like structure, usually 4-6 A root-mean-square deviation from the native, is located. For beta-proteins larger than 75 residues, the energy gap between native-like structures and the lowest energy structures produced in the simulation is large, so that low RMSD structures are not generated starting from an unfolded state. This is attributed to the lack of an explicit hydrogen bond term in the potential function, which we hypothesize is necessary to stabilize large assemblies of beta-strands. Copyright 1999 Academic Press.

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats.

PubMed

Frigolet, Maria E; Torres, Nimbe; Uribe-Figueroa, Laura; Rangel, Claudia; Jimenez-Sanchez, Gerardo; Tovar, Armando R

2011-02-01

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042

PubMed Central

2018-01-01

ABSTRACT Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae. IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool. PMID:29577085

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042.

PubMed

Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

2018-01-01

Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

The planetary biology of cytochrome P450 aromatases.

PubMed

Gaucher, Eric A; Graddy, Logan G; Li, Tang; Simmen, Rosalia C M; Simmen, Frank A; Schreiber, David R; Liberles, David A; Janis, Christine M; Benner, Steven A

2004-08-17

Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system. Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

The planetary biology of cytochrome P450 aromatases

PubMed Central

Gaucher, Eric A; Graddy, Logan G; Li, Tang; Simmen, Rosalia CM; Simmen, Frank A; Schreiber, David R; Liberles, David A; Janis, Christine M; Benner, Steven A

2004-01-01

Background Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system. Results Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases–enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. Conclusions This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems. PMID:15315709

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

PubMed Central

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed Central

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-01-01

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart. Images Fig. 1 Fig. 3 PMID:8637874

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

PubMed

Murata, Michio; Sugiyama, Shigeru; Matsuoka, Shigeru; Matsumori, Nobuaki

2015-08-01

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. © 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Challenges and perspectives of metaproteomic data analysis.

PubMed

Heyer, Robert; Schallert, Kay; Zoun, Roman; Becher, Beatrice; Saake, Gunter; Benndorf, Dirk

2017-11-10

In nature microorganisms live in complex microbial communities. Comprehensive taxonomic and functional knowledge about microbial communities supports medical and technical application such as fecal diagnostics as well as operation of biogas plants or waste water treatment plants. Furthermore, microbial communities are crucial for the global carbon and nitrogen cycle in soil and in the ocean. Among the methods available for investigation of microbial communities, metaproteomics can approximate the activity of microorganisms by investigating the protein content of a sample. Although metaproteomics is a very powerful method, issues within the bioinformatic evaluation impede its success. In particular, construction of databases for protein identification, grouping of redundant proteins as well as taxonomic and functional annotation pose big challenges. Furthermore, growing amounts of data within a metaproteomics study require dedicated algorithms and software. This review summarizes recent metaproteomics software and addresses the introduced issues in detail. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Protein domain organisation: adding order.

PubMed

Kummerfeld, Sarah K; Teichmann, Sarah A

2009-01-29

Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and reverse orientation in different proteins relative to random graphs with identical degree distributions. While these features were statistically over-represented, they are still fairly rare. Looking in detail at the proteins involved, we found strong functional relationships within each cluster. In addition, the domains tended to be involved in protein-protein interaction and are able to function as independent structural units. A particularly striking example was the human Jak-STAT signalling pathway which makes use of a set of domains in a range of orders and orientations to provide nuanced signaling functionality. This illustrated the importance of functional and structural constraints (or lack thereof) on domain organisation.

Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis

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

Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.

2011-02-17

Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular {alpha}-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins ({approx}99%) with known functions were expressed, whereas only approximately 80% of hypothetical proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in bothmore » bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.« less

Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.

PubMed

Peng, Hui; Zhang, Yixiang; Palmer, Lauren D; Kehl-Fie, Thomas E; Skaar, Eric P; Trinidad, Jonathan C; Giedroc, David P

2017-10-13

Hydrogen sulfide (H 2 S) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of H 2 S signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in H 2 S signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that H 2 S signaling can be sensed by global regulators to affect the expression of virulence factors.

Global transcriptome analysis of eukaryotic genes affected by gromwell extract.

PubMed

Bang, Soohyun; Lee, Dohyun; Kim, Hanhe; Park, Jiyong; Bahn, Yong-Sun

2014-02-01

Gromwell is known to have diverse pharmacological, cosmetic and nutritional benefits for humans. Nevertheless, the biological influence of gromwell extract (GE) on the general physiology of eukaryotic cells remains unknown. In this study a global transcriptome analysis was performed to identify genes affected by the addition of GE with Cryptococcus neoformans as the model system. In response to GE treatment, genes involved in signal transduction were immediately regulated, and the evolutionarily conserved sets of genes involved in the core cellular functions, including DNA replication, RNA transcription/processing and protein translation/processing, were generally up-regulated. In contrast, a number of genes involved in carbohydrate metabolism and transport, inorganic ion transport and metabolism, post-translational modification/protein turnover/chaperone functions and signal transduction were down-regulated. Among the GE-responsive genes that are also evolutionarily conserved in the human genome, the expression patterns of YSA1, TPO2, CFO1 and PZF1 were confirmed by northern blot analysis. Based on the functional characterization of some GE-responsive genes, it was found that GE treatment may promote cellular tolerance against a variety of environmental stresses in eukaryotes. GE treatment affects the expression levels of a significant portion of the Cryptococcus genome, implying that GE significantly affects the general physiology of eukaryotic cells. © 2013 Society of Chemical Industry.

Massive expansion and differential evolution of small heat shock proteins with wheat (Triticum aestivum L.) polyploidization.

PubMed

Wang, Xiaoming; Wang, Ruochen; Ma, Chuang; Shi, Xue; Liu, Zhenshan; Wang, Zhonghua; Sun, Qixin; Cao, Jun; Xu, Shengbao

2017-05-31

Wheat (Triticum aestivum), one of the world's most important crops, is facing unprecedented challenges due to global warming. To evaluate the gene resources for heat adaptation in hexaploid wheat, small heat shock proteins (sHSPs), the key plant heat protection genes, were comprehensively analysed in wheat and related species. We found that the sHSPs of hexaploid wheat were massively expanded in A and B subgenomes with intrachromosomal duplications during polyploidization. These expanded sHSPs were under similar purifying selection and kept the expressional patterns with the original copies. Generally, a strong purifying selection acted on the α-crystallin domain (ACD) and theoretically constrain conserved function. Meanwhile, weaker purifying selection and strong positive selection acted on the N-terminal region, which conferred sHSP flexibility, allowing adjustments to a wider range of substrates in response to genomic and environmental changes. Notably, in CI, CV, ER, MI and MII subfamilies, gene duplications, expression variations and functional divergence occurred before wheat polyploidization. Our results indicate the massive expansion of active sHSPs in hexaploid wheat may also provide more raw materials for evolving functional novelties and generating genetic diversity to face future global climate changes, and highlight the expansion of stress response genes with wheat polyploidization.

High-confidence prediction of global interactomes based on genome-wide coevolutionary networks

PubMed Central

Juan, David; Pazos, Florencio; Valencia, Alfonso

2008-01-01

Interacting or functionally related protein families tend to have similar phylogenetic trees. Based on this observation, techniques have been developed to predict interaction partners. The observed degree of similarity between the phylogenetic trees of two proteins is the result of many different factors besides the actual interaction or functional relationship between them. Such factors influence the performance of interaction predictions. One aspect that can influence this similarity is related to the fact that a given protein interacts with many others, and hence it must adapt to all of them. Accordingly, the interaction or coadaptation signal within its tree is a composite of the influence of all of the interactors. Here, we introduce a new estimator of coevolution to overcome this and other problems. Instead of relying on the individual value of tree similarity between two proteins, we use the whole network of similarities between all of the pairs of proteins within a genome to reassess the similarity of that pair, thereby taking into account its coevolutionary context. We show that this approach offers a substantial improvement in interaction prediction performance, providing a degree of accuracy/coverage comparable with, or in some cases better than, that of experimental techniques. Moreover, important information on the structure, function, and evolution of macromolecular complexes can be inferred with this methodology. PMID:18199838

High-confidence prediction of global interactomes based on genome-wide coevolutionary networks.

PubMed

Juan, David; Pazos, Florencio; Valencia, Alfonso

2008-01-22

Interacting or functionally related protein families tend to have similar phylogenetic trees. Based on this observation, techniques have been developed to predict interaction partners. The observed degree of similarity between the phylogenetic trees of two proteins is the result of many different factors besides the actual interaction or functional relationship between them. Such factors influence the performance of interaction predictions. One aspect that can influence this similarity is related to the fact that a given protein interacts with many others, and hence it must adapt to all of them. Accordingly, the interaction or coadaptation signal within its tree is a composite of the influence of all of the interactors. Here, we introduce a new estimator of coevolution to overcome this and other problems. Instead of relying on the individual value of tree similarity between two proteins, we use the whole network of similarities between all of the pairs of proteins within a genome to reassess the similarity of that pair, thereby taking into account its coevolutionary context. We show that this approach offers a substantial improvement in interaction prediction performance, providing a degree of accuracy/coverage comparable with, or in some cases better than, that of experimental techniques. Moreover, important information on the structure, function, and evolution of macromolecular complexes can be inferred with this methodology.

Oxidative stress-triggered interactions between the succinyl- and acetyl-proteomes of rice leaves.

PubMed

Zhou, Heng; Finkemeier, Iris; Guan, Wenxue; Tossounian, Maria-Armineh; Wei, Bo; Young, David; Huang, Jingjing; Messens, Joris; Yang, Xibin; Zhu, Jun; Wilson, Michael H; Shen, Wenbiao; Xie, Yanjie; Foyer, Christine H

2018-05-01

Protein lysine acylations, such as succinylation and acetylation, are important post-translational modification (PTM) mechanisms, with key roles in cellular regulation. Antibody-based affinity enrichment, high-resolution liquid chromatography mass spectrometry analysis, and integrated bioinformatics analysis were used to characterize the lysine succinylome (K suc ) and acetylome (K ace ) of rice leaves. In total, 2,593 succinylated and 1,024 acetylated proteins were identified, of which 723 were simultaneously acetylated and succinylated. Proteins involved in photosynthetic carbon metabolism such as the large and small subunits of RuBisCO, ribosomal functions, and other key processes were subject to both PTMs. Preliminary insights into oxidant-induced changes to the rice acetylome and succinylome were gained from treatments with hydrogen peroxide. Exposure to oxidative stress did not regulate global changes in the rice acetylome or succinylome but rather led to modifications on a specific subset of the identified sites. De-succinylation of recombinant catalase (CATA) and glutathione S-transferase (OsGSTU6) altered the activities of these enzymes showing that this PTM may have a regulatory function. These findings not only greatly extend the list of acetylated and/or succinylated proteins but they also demonstrate the close cooperation between these PTMs in leaf proteins with key metabolic functions. © 2017 John Wiley & Sons Ltd.

Transduction of anti-cell death protein FNK protects isolated rat hearts from myocardial infarction induced by ischemia/reperfusion.

PubMed

Arakawa, Masayuki; Yasutake, Masahiro; Miyamoto, Masaaki; Takano, Teruo; Asoh, Sadamitsu; Ohta, Shigeo

2007-05-08

Artificial anti-cell death protein FNK, a Bcl-x(L) derivative with three amino acid-substitutions (Y22F, Q26N, and R165K) has enhanced anti-apoptotic and anti-necrotic activity and facilitates cell survival in many species and cell types. The objectives of this study were (i) to investigate whether the protein conjugated with a protein transduction domain (PTD-FNK) reduces myocardial infarct size and improves post-ischemic cardiac function in ischemic/reperfused rat hearts, and (ii) to understand the mechanism(s) by which PTD-FNK exerts a protective effect. Isolated rat hearts were subjected to 35-min global ischemia, followed by 120-min reperfusion using the Langendorff methods. PTD-FNK (a total of 30 microl) was injected intramuscularly into the anterior wall of the left ventricle either at 1 min after induction of global ischemia (group A) or at 30 min after induction of global ischemia (at 5 min before reperfusion) (group B). In group A, infarct size was significantly reduced from 47.8+/-6.8% in the control to 30.4+/-5.2, 28.7+/-3.8, and 30.4+/-6.8% with PTD-FNK at 5, 50, and 500 nmol/l, respectively (p<0.05). Temporal recovery of left ventricular developed pressure at 60 min and 120 min after reperfusion was significantly better in PTD-FNK (50 and 500 nmol/l)-treated groups than in the control (p<0.05). In contrast, PTD-FNK treatment had no effect on group B. Western blot analysis showed that PTD-FNK markedly inhibited procaspase-3 cleavage (activation of caspase-3) and reduced the number of nuclei stained by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphoshate nick-end labeling (TUNEL) assay. These findings suggest that PTD-FNK reduces the volume of myocardial infarction with corresponding functional recovery, at least in part, through the suppression of myocardial apoptosis following ischemia/reperfusion.

Screened exchange hybrid density functional for accurate and efficient structures and interaction energies.

PubMed

Brandenburg, Jan Gerit; Caldeweyher, Eike; Grimme, Stefan

2016-06-21

We extend the recently introduced PBEh-3c global hybrid density functional [S. Grimme et al., J. Chem. Phys., 2015, 143, 054107] by a screened Fock exchange variant based on the Henderson-Janesko-Scuseria exchange hole model. While the excellent performance of the global hybrid is maintained for small covalently bound molecules, its performance for computed condensed phase mass densities is further improved. Most importantly, a speed up of 30 to 50% can be achieved and especially for small orbital energy gap cases, the method is numerically much more robust. The latter point is important for many applications, e.g., for metal-organic frameworks, organic semiconductors, or protein structures. This enables an accurate density functional based electronic structure calculation of a full DNA helix structure on a single core desktop computer which is presented as an example in addition to comprehensive benchmark results.

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

Kolker, Eugene

Our project focused primarily on analysis of different types of data produced by global high-throughput technologies, data integration of gene annotation, and gene and protein expression information, as well as on getting a better functional annotation of Shewanella genes. Specifically, four of our numerous major activities and achievements include the development of: statistical models for identification and expression proteomics, superior to currently available approaches (including our own earlier ones); approaches to improve gene annotations on the whole-organism scale; standards for annotation, transcriptomics and proteomics approaches; and generalized approaches for data integration of gene annotation, gene and protein expression information.

RNA silencing in plant symbiotic bacteria: Insights from a protein-centric view.

PubMed

Jiménez-Zurdo, José I; Robledo, Marta

2017-12-02

Extensive work in model enterobacteria has evidenced that the RNA chaperone Hfq and several endoribonucleases, such as RNase E or RNase III, serve pivotal roles in small RNA-mediated post-transcriptional silencing of gene expression. Characterization of these protein hubs commonly provide global functional and mechanistic insights into complex sRNA regulatory networks. The legume endosymbiont Sinorhizobium meliloti is a non-classical model bacterium with a very complex lifestyle in which riboregulation is expected to play important adaptive functions. Here, we discuss current knowledge about RNA silencing in S. meliloti from the perspective of the activity of Hfq and a recently discovered endoribonuclease (YbeY) exhibiting unprecedented catalytic versatility for the cleavage of single- and double-stranded RNA molecules.

O-GlcNAc cycling in the developing, adult and geriatric brain.

PubMed

Lagerlöf, Olof

2018-06-01

Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.

Quantification of Cysteinyl-S-Nitrosylation by Fluorescence in Unbiased Proteomic Studies*

PubMed Central

Wiktorowicz, John E.; Stafford, Susan; Rea, Harriet; Urvil, Petri; Soman, Kizhake; Kurosky, Alexander; Perez-Polo, J. Regino; Savidge, Tor C.

2011-01-01

Cysteinyl-S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins due to physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia reperfusion model, and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique’s power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses. PMID:21615140

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias).

PubMed

Lee, Jinoo; Valkova, Nelly; White, Mark P; Kültz, Dietmar

2006-09-01

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.

Unified Alignment of Protein-Protein Interaction Networks.

PubMed

Malod-Dognin, Noël; Ban, Kristina; Pržulj, Nataša

2017-04-19

Paralleling the increasing availability of protein-protein interaction (PPI) network data, several network alignment methods have been proposed. Network alignments have been used to uncover functionally conserved network parts and to transfer annotations. However, due to the computational intractability of the network alignment problem, aligners are heuristics providing divergent solutions and no consensus exists on a gold standard, or which scoring scheme should be used to evaluate them. We comprehensively evaluate the alignment scoring schemes and global network aligners on large scale PPI data and observe that three methods, HUBALIGN, L-GRAAL and NATALIE, regularly produce the most topologically and biologically coherent alignments. We study the collective behaviour of network aligners and observe that PPI networks are almost entirely aligned with a handful of aligners that we unify into a new tool, Ulign. Ulign enables complete alignment of two networks, which traditional global and local aligners fail to do. Also, multiple mappings of Ulign define biologically relevant soft clusterings of proteins in PPI networks, which may be used for refining the transfer of annotations across networks. Hence, PPI networks are already well investigated by current aligners, so to gain additional biological insights, a paradigm shift is needed. We propose such a shift come from aligning all available data types collectively rather than any particular data type in isolation from others.

Unveiling network-based functional features through integration of gene expression into protein networks.

PubMed

Jalili, Mahdi; Gebhardt, Tom; Wolkenhauer, Olaf; Salehzadeh-Yazdi, Ali

2018-06-01

Decoding health and disease phenotypes is one of the fundamental objectives in biomedicine. Whereas high-throughput omics approaches are available, it is evident that any single omics approach might not be adequate to capture the complexity of phenotypes. Therefore, integrated multi-omics approaches have been used to unravel genotype-phenotype relationships such as global regulatory mechanisms and complex metabolic networks in different eukaryotic organisms. Some of the progress and challenges associated with integrated omics studies have been reviewed previously in comprehensive studies. In this work, we highlight and review the progress, challenges and advantages associated with emerging approaches, integrating gene expression and protein-protein interaction networks to unravel network-based functional features. This includes identifying disease related genes, gene prioritization, clustering protein interactions, developing the modules, extract active subnetworks and static protein complexes or dynamic/temporal protein complexes. We also discuss how these approaches contribute to our understanding of the biology of complex traits and diseases. This article is part of a Special Issue entitled: Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of the novel algorithm of flexible ligand docking with moveable target-protein atoms.

PubMed

Sulimov, Alexey V; Zheltkov, Dmitry A; Oferkin, Igor V; Kutov, Danil C; Katkova, Ekaterina V; Tyrtyshnikov, Eugene E; Sulimov, Vladimir B

2017-01-01

We present the novel docking algorithm based on the Tensor Train decomposition and the TT-Cross global optimization. The algorithm is applied to the docking problem with flexible ligand and moveable protein atoms. The energy of the protein-ligand complex is calculated in the frame of the MMFF94 force field in vacuum. The grid of precalculated energy potentials of probe ligand atoms in the field of the target protein atoms is not used. The energy of the protein-ligand complex for any given configuration is computed directly with the MMFF94 force field without any fitting parameters. The conformation space of the system coordinates is formed by translations and rotations of the ligand as a whole, by the ligand torsions and also by Cartesian coordinates of the selected target protein atoms. Mobility of protein and ligand atoms is taken into account in the docking process simultaneously and equally. The algorithm is realized in the novel parallel docking SOL-P program and results of its performance for a set of 30 protein-ligand complexes are presented. Dependence of the docking positioning accuracy is investigated as a function of parameters of the docking algorithm and the number of protein moveable atoms. It is shown that mobility of the protein atoms improves docking positioning accuracy. The SOL-P program is able to perform docking of a flexible ligand into the active site of the target protein with several dozens of protein moveable atoms: the native crystallized ligand pose is correctly found as the global energy minimum in the search space with 157 dimensions using 4700 CPU ∗ h at the Lomonosov supercomputer.

Enlazin, a Natural Fusion of Two Classes of Canonical Cytoskeletal Proteins, Contributes to Cytokinesis Dynamics

PubMed Central

Octtaviani, Edelyn; Effler, Janet C.

2006-01-01

Cytokinesis requires a complex network of equatorial and global proteins to regulate cell shape changes. Here, using interaction genetics, we report the first characterization of a novel protein, enlazin. Enlazin is a natural fusion of two canonical classes of actin-associated proteins, the ezrin-radixin-moesin family and fimbrin, and it is localized to actin-rich structures. A fragment of enlazin, enl-tr, was isolated as a genetic suppressor of the cytokinesis defect of cortexillin-I mutants. Expression of enl-tr disrupts expression of endogenous enlazin, indicating that enl-tr functions as a dominant-negative lesion. Enlazin is distributed globally during cytokinesis and is required for cortical tension and cell adhesion. Consistent with a role in cell mechanics, inhibition of enlazin in a cortexillin-I background restores cytokinesis furrowing dynamics and suppresses the growth-in-suspension defect. However, as expected for a role in cell adhesion, inhibiting enlazin in a myosin-II background induces a synthetic cytokinesis phenotype, frequently arresting furrow ingression at the dumbbell shape and/or causing recession of the furrow. Thus, enlazin has roles in cell mechanics and adhesion, and these roles seem to be differentially significant for cytokinesis, depending on the genetic background. PMID:17050732

Studies of molecular interactions between beta-lactoglobulin and sugar beet pectin at neutral pH by high performance size exclusion chromatography

USDA-ARS?s Scientific Manuscript database

Foods that are rich in protein and other micronutrients such as bioactives and vitamins but low in sugar and fat may help mitigate the global obesity epidemic. Specialty ingredients can be fabricated to possess specific functions such as encapsulating bioactives through controlled assembly of protei...

Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

PubMed

Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

2008-11-15

Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions.

Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

PubMed

Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

2016-06-03

Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria.

The potential impact of animal protein intake on global and abdominal obesity: evidence from the Observation of Cardiovascular Risk Factors in Luxembourg (ORISCAV-LUX) study.

PubMed

Alkerwi, Ala'a; Sauvageot, Nicolas; Buckley, Jonathan D; Donneau, Anne-Françoise; Albert, Adelin; Guillaume, Michèle; Crichton, Georgina E

2015-07-01

To examine the association of total animal protein intake and protein derived from different dietary sources (meat; fish and shellfish; eggs; milk products) with global and abdominal obesity among adults in Luxembourg. Binary logistic regression analysis was used to assess the relationship between animal protein intake (as a percentage of total energy intake) and global obesity (BMI ≥ 30.0 kg/m(2)) and abdominal obesity (waist circumference ≥ 102 cm for men and ≥ 88 cm for women), after controlling for potential confounders. Observation of Cardiovascular Risk Factors in Luxembourg (ORISCAV-LUX) study. The study population was derived from a national cross-sectional stratified sample of 1152 individuals aged 18-69 years, recruited between November 2007 and January 2009. There was an independent positive association between total animal protein intake and both global (OR = 1.18; 95% CI 1.12, 1.25) and abdominal obesity (OR = 1.14; 95% CI 1.08, 1.20) after adjustment for age, gender, education, smoking, physical activity and intakes of total fat, carbohydrate, fibre, and fruit and vegetables. Protein intakes from meat, fish and shellfish were positively associated with global and abdominal obesity with further adjustment for vegetal protein and other sources of animal-derived protein (all P < 0.01). Protein derived from eggs or milk products was unrelated to global or abdominal obesity. Our findings suggest that protein derived from animal sources, in particular from meat, fish and shellfish, may be associated with increased risk of both global and abdominal obesity among presumably healthy adults in Luxembourg. These findings suggest that lower animal protein intakes may be important for maintenance of healthy body weight.

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.

A large dataset of protein dynamics in the mammalian heart proteome

PubMed Central

Lau, Edward; Cao, Quan; Ng, Dominic C.M.; Bleakley, Brian J.; Dincer, T. Umut; Bot, Brian M.; Wang, Ding; Liem, David A.; Lam, Maggie P.Y.; Ge, Junbo; Ping, Peipei

2016-01-01

Protein stability is a major regulatory principle of protein function and cellular homeostasis. Despite limited understanding on mechanisms, disruption of protein turnover is widely implicated in diverse pathologies from heart failure to neurodegenerations. Information on global protein dynamics therefore has the potential to expand the depth and scope of disease phenotyping and therapeutic strategies. Using an integrated platform of metabolic labeling, high-resolution mass spectrometry and computational analysis, we report here a comprehensive dataset of the in vivo half-life of 3,228 and the expression of 8,064 cardiac proteins, quantified under healthy and hypertrophic conditions across six mouse genetic strains commonly employed in biomedical research. We anticipate these data will aid in understanding key mitochondrial and metabolic pathways in heart diseases, and further serve as a reference for methodology development in dynamics studies in multiple organ systems. PMID:26977904

CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function

PubMed Central

Krüger, Dennis M.; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

2013-01-01

The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein’s (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement. PMID:23609541

ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

PubMed Central

Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai

2001-01-01

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164

Global-scale profiling of differential expressed lysine acetylated proteins in colorectal cancer tumors and paired liver metastases.

PubMed

Shen, Zhanlong; Wang, Bo; Luo, Jianyuan; Jiang, Kewei; Zhang, Hui; Mustonen, Harri; Puolakkainen, Pauli; Zhu, Jun; Ye, Yingjiang; Wang, Shan

2016-06-16

Lysine acetylated modification was indicated to impact colorectal cancer (CRC)'s distant metastasis. However, the global acetylated proteins in CRC and the differential expressed acetylated proteins and acetylated sites between CRC primary and distant metastatic tumor remains unclear. Our aim was to construct a complete atlas of acetylome in CRC and paired liver metastases. Combining high affinity enrichment of acetylated peptides with high sensitive mass spectrometry, we identified 603 acetylation sites from 316 proteins, among which 462 acetylation sites corresponding to 243 proteins were quantified. We further classified them into groups according to cell component, molecular function and biological process and analyzed the metabolic pathways, domain structures and protein interaction networks. Finally, we evaluated the differentially expressed lysine acetylation sites and revealed that 31 acetylated sites of 22 proteins were downregulated in CRC liver metastases compared to that in primary CRC while 40 acetylated sites of 32 proteins were upregulated, of which HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. These results provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. This study described provides, for the first time, that full-scale profiling of lysine acetylated proteins were identified and quantified in colorectal cancer (CRC) and paired liver metastases. The novelty of the study is that we constructed a complete atlas of acetylome in CRC and paired liver metastases. Moreover, we analyzed these differentially expressed acetylated proteins in cell component, molecular function and biological process. In addition, metabolic pathways, domain structures and protein interaction networks of acetylated proteins were also investigated. Our approaches shows that of the differentially expressed proteins, HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. Our findings provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes

PubMed Central

Hwang, Hyonson; Bowen, Benjamin P.; Lefort, Natalie; Flynn, Charles R.; De Filippis, Elena A.; Roberts, Christine; Smoke, Christopher C.; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J.

2010-01-01

OBJECTIVE Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here. RESEARCH DESIGN AND METHODS Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors. RESULTS Of 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased). CONCLUSIONS The results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance. PMID:19833877

Aggregation of alpha-synuclein by a coarse-grained Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Farmer, Barry; Pandey, Ras

Alpha-synuclein, an intrinsic protein abundant in neurons, is believed to be a major cause of neurodegenerative diseases (e.g. Alzheimer, Parkinson's disease). Abnormal aggregation of ASN leads to Lewy bodies with specific morphologies. We investigate the self-organizing structures in a crowded environment of ASN proteins by a coarse-grained Monte Carlo simulation. ASN is a chain of 140 residues. Structure detail of residues is neglected but its specificity is captured via unique knowledge-based residue-residue interactions. Large-scale simulations are performed to analyze a number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) as a function of temperature and protein concentration. Trend in multi-scale structural variations of the protein in a crowded environment is compared with that of a free protein chain.

Thrombin generation, ProC(®)Global, prothrombin time and activated partial thromboplastin time in thawed plasma stored for seven days and after methylene blue/light pathogen inactivation.

PubMed

Thiele, Thomas; Hron, Gregor; Kellner, Sarah; Wasner, Christina; Westphal, Antje; Warkentin, Theodore E; Greinacher, Andreas; Selleng, Kathleen

2016-01-01

Methylene blue pathogen inactivation and storage of thawed plasma both lead to changes in the activity of several clotting factors. We investigated how this translates into a global loss of thrombin generation potential and alterations in the protein C pathway. Fifty apheresis plasma samples were thawed and each divided into three subunits. One subunit was stored for 7 days at 4 °C, one was stored for 7 days at 22 °C and one was stored at 4 °C after methylene blue/light treatment. Thrombin generation parameters, ProC(®)Global-NR, prothrombin time and activated partial thromboplastin time were assessed on days 0 and 7. The velocity of thrombin generation increased significantly after methylene blue treatment (increased thrombin generation rate; time to peak decreased) and decreased after storage (decreased thrombin generation rate and peak thrombin; increased lag time and time to peak). The endogenous thrombin generation potential remained stable after methylene blue treatment and storage at 4 °C. Methylene blue treatment and 7 days of storage at 4 °C activated the protein C pathway, whereas storage at room temperature and storage after methylene blue treatment decreased the functional capacity of the protein C pathway. Prothrombin time and activated partial thromboplastin time showed only modest alterations. The global clotting capacity of thawed plasma is maintained at 4 °C for 7 days and directly after methylene blue treatment of thawed plasma. Thrombin generation and ProC(®)Global are useful tools for investigating the impact of pathogen inactivation and storage on the clotting capacity of therapeutic plasma preparations.

Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

PubMed

Yan, Shao-Min; Wu, Guang

2009-12-01

The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

PubMed Central

Yao, Chunxiang; Behring, Jessica B.; Shao, Di; Sverdlov, Aaron L.; Whelan, Stephen A.; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A.; Seta, Francesca; Costello, Catherine E.; Cohen, Richard A.; Matsui, Reiko; Colucci, Wilson S.; McComb, Mark E.; Bachschmid, Markus M.

2015-01-01

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

PubMed

Tian, Ruijun; Jin, Jing; Taylor, Lorne; Larsen, Brett; Quaggin, Susan E; Pawson, Tony

2013-04-01

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

PubMed

Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G

2002-08-01

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

PubMed

Geng, Xiaofang; Wang, Gaiping; Qin, Yanli; Zang, Xiayan; Li, Pengfei; Geng, Zhi; Xue, Deming; Dong, Zimei; Ma, Kexue; Chen, Guangwen; Xu, Cunshuan

2015-01-01

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae

PubMed Central

Paulo, Joao A.; O’Connell, Jeremy D.; Gaun, Aleksandr; Gygi, Steven P.

2015-01-01

The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry–based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid–related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production. PMID:26399295

Protein social behavior makes a stronger signal for partner identification than surface geometry.

PubMed

Laine, Elodie; Carbone, Alessandra

2017-01-01

Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico-chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross-docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S-index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface-based (ranking) score to discriminate partners from non-interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137-154. © 2016 Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

PDB2Graph: A toolbox for identifying critical amino acids map in proteins based on graph theory.

PubMed

Niknam, Niloofar; Khakzad, Hamed; Arab, Seyed Shahriar; Naderi-Manesh, Hossein

2016-05-01

The integrative and cooperative nature of protein structure involves the assessment of topological and global features of constituent parts. Network concept takes complete advantage of both of these properties in the analysis concomitantly. High compatibility to structural concepts or physicochemical properties in addition to exploiting a remarkable simplification in the system has made network an ideal tool to explore biological systems. There are numerous examples in which different protein structural and functional characteristics have been clarified by the network approach. Here, we present an interactive and user-friendly Matlab-based toolbox, PDB2Graph, devoted to protein structure network construction, visualization, and analysis. Moreover, PDB2Graph is an appropriate tool for identifying critical nodes involved in protein structural robustness and function based on centrality indices. It maps critical amino acids in protein networks and can greatly aid structural biologists in selecting proper amino acid candidates for manipulating protein structures in a more reasonable and rational manner. To introduce the capability and efficiency of PDB2Graph in detail, the structural modification of Calmodulin through allosteric binding of Ca(2+) is considered. In addition, a mutational analysis for three well-identified model proteins including Phage T4 lysozyme, Barnase and Ribonuclease HI, was performed to inspect the influence of mutating important central residues on protein activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioactive Peptides Derived from Seaweed Protein and Their Health Benefits: Antihypertensive, Antioxidant, and Antidiabetic Properties.

PubMed

Admassu, Habtamu; Gasmalla, Mohammed Abdalbasit A; Yang, Ruijin; Zhao, Wei

2018-01-01

Cardiovascular diseases and diabetes are the biggest causes of death globally. Therefore, prevention of these diseases is a focus of pharmaceuticals and functional food manufacturers. This review summarizes recent research trends and scientific knowledge in seaweed protein-derived peptides with particular emphasis on production, isolation and potential health impacts in prevention of hypertension, diabetes and oxidative stress. The current status and future prospects of bioactive peptides are also discussed. Bioactive peptides have strong potential for use in therapeutic drug and functional food formulation in health management strategy, especially cardiovascular disease and diabetes. Seaweeds can be used as sustainable protein sources in the production of these peptide-based drugs and functional foods for preventing such diseases. Many studies have reported that peptides showing angiotensin converting enzyme inhibition, antihypertensive, antioxidative and antidiabetics activities, have been successfully isolated from seaweed. However, further research is needed in large-scale production of these peptides, efficient isolation methods, interactions with functional foods and other pharmaceuticals, and their ease to digestion in in vivo studies and safety to validate the health benefits of these peptides. © 2017 Institute of Food Technologists®.

Structure based alignment and clustering of proteins (STRALCP)

DOEpatents

Zemla, Adam T.; Zhou, Carol E.; Smith, Jason R.; Lam, Marisa W.

2013-06-18

Disclosed are computational methods of clustering a set of protein structures based on local and pair-wise global similarity values. Pair-wise local and global similarity values are generated based on pair-wise structural alignments for each protein in the set of protein structures. Initially, the protein structures are clustered based on pair-wise local similarity values. The protein structures are then clustered based on pair-wise global similarity values. For each given cluster both a representative structure and spans of conserved residues are identified. The representative protein structure is used to assign newly-solved protein structures to a group. The spans are used to characterize conservation and assign a "structural footprint" to the cluster.

Gene regulatory networks in lactation: identification of global principles using bioinformatics.

PubMed

Lemay, Danielle G; Neville, Margaret C; Rudolph, Michael C; Pollard, Katherine S; German, J Bruce

2007-11-27

The molecular events underlying mammary development during pregnancy, lactation, and involution are incompletely understood. Mammary gland microarray data, cellular localization data, protein-protein interactions, and literature-mined genes were integrated and analyzed using statistics, principal component analysis, gene ontology analysis, pathway analysis, and network analysis to identify global biological principles that govern molecular events during pregnancy, lactation, and involution. Several key principles were derived: (1) nearly a third of the transcriptome fluctuates to build, run, and disassemble the lactation apparatus; (2) genes encoding the secretory machinery are transcribed prior to lactation; (3) the diversity of the endogenous portion of the milk proteome is derived from fewer than 100 transcripts; (4) while some genes are differentially transcribed near the onset of lactation, the lactation switch is primarily post-transcriptionally mediated; (5) the secretion of materials during lactation occurs not by up-regulation of novel genomic functions, but by widespread transcriptional suppression of functions such as protein degradation and cell-environment communication; (6) the involution switch is primarily transcriptionally mediated; and (7) during early involution, the transcriptional state is partially reverted to the pre-lactation state. A new hypothesis for secretory diminution is suggested - milk production gradually declines because the secretory machinery is not transcriptionally replenished. A comprehensive network of protein interactions during lactation is assembled and new regulatory gene targets are identified. Less than one fifth of the transcriptionally regulated nodes in this lactation network have been previously explored in the context of lactation. Implications for future research in mammary and cancer biology are discussed.

A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification.

PubMed

Shen, Bingquan; Zhang, Wanjun; Shi, Zhaomei; Tian, Fang; Deng, Yulin; Sun, Changqing; Wang, Guangshun; Qin, Weijie; Qian, Xiaohong

2017-07-01

O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine. Copyright © 2017. Published by Elsevier B.V.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

PubMed

Mahadevan, Vivek; Khademullah, C Sahara; Dargaei, Zahra; Chevrier, Jonah; Uvarov, Pavel; Kwan, Julian; Bagshaw, Richard D; Pawson, Tony; Emili, Andrew; De Koninck, Yves; Anggono, Victor; Airaksinen, Matti; Woodin, Melanie A

2017-10-13

KCC2 is a neuron-specific K + -Cl - cotransporter essential for establishing the Cl - gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl - . Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

PubMed Central

Mahadevan, Vivek; Chevrier, Jonah; Uvarov, Pavel; Kwan, Julian; Bagshaw, Richard D; Pawson, Tony; Emili, Andrew; De Koninck, Yves; Anggono, Victor; Airaksinen, Matti

2017-01-01

KCC2 is a neuron-specific K+-Cl– cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2. PMID:29028184

Ensemble-Based Computational Approach Discriminates Functional Activity of p53 Cancer and Rescue Mutants

PubMed Central

Demir, Özlem; Baronio, Roberta; Salehi, Faezeh; Wassman, Christopher D.; Hall, Linda; Hatfield, G. Wesley; Chamberlin, Richard; Kaiser, Peter; Lathrop, Richard H.; Amaro, Rommie E.

2011-01-01

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r2 = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants. PMID:22028641

Antennal proteome comparison of sexually mature drone and forager honeybees.

PubMed

Feng, Mao; Song, Feifei; Aleku, Dereje Woltedji; Han, Bin; Fang, Yu; Li, Jianke

2011-07-01

Honeybees have evolved an intricate system of chemical communication to regulate their complex social interactions. Specific proteins involved in odorant detection most likely supported this chemical communication. Odorant reception takes place mainly in the antennae within hairlike structures called olfactory sensilla. Antennal proteomes of sexually mature drone and forager worker bees (an age group of bees assigned to perform field tasks) were compared using two-dimensional electrophoresis, mass spectrometry, quantitative real-time polymerase chain reaction, and bioinformatics. Sixty-one differentially expressed proteins were identified in which 67% were highly upregulated in the drones' antennae whereas only 33% upregulated in the worker bees' antennae. The antennae of the worker bees strongly expressed carbohydrate and energy metabolism and molecular transporters signifying a strong demand for metabolic energy and odorant binding proteins for their foraging activities and other olfactory responses, while proteins related to fatty acid metabolism, antioxidation, and protein folding were strongly upregulated in the drones' antennae as an indication of the importance for the detection and degradation of sex pheromones during queen identification for mating. On the basis of both groups of altered antenna proteins, carbohydrate metabolism and energy production and molecular transporters comprised more than 80% of the functional enrichment analysis and 45% of the constructed biological interaction networks (BIN), respectively. This suggests these two protein families play crucial roles in the antennal olfactory function of sexually mature drone and forager worker bees. Several key node proteins in the BIN were validated at the transcript level. This first global proteomic comparative analysis of antennae reveals sex-biased protein expression in both bees, indicating that odorant response mechanisms are sex-specific because of natural selection for different olfactory functions. To the best of our knowledge, this result further provides extensive insight into the expression of the proteins in the antennae of drone and worker honeybees and adds vital information to the previous findings. It also provides a new angle for future detailed functional analysis of the antennae of the honeybee castes.

Global functional analyses of cellular responses to pore-forming toxins.

PubMed

Kao, Cheng-Yuan; Los, Ferdinand C O; Huffman, Danielle L; Wachi, Shinichiro; Kloft, Nicole; Husmann, Matthias; Karabrahimi, Valbona; Schwartz, Jean-Louis; Bellier, Audrey; Ha, Christine; Sagong, Youn; Fan, Hui; Ghosh, Partho; Hsieh, Mindy; Hsu, Chih-Shen; Chen, Li; Aroian, Raffi V

2011-03-01

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

TULIPs: tunable, light-controlled interacting protein tags for cell biology.

PubMed

Strickland, Devin; Lin, Yuan; Wagner, Elizabeth; Hope, C Matthew; Zayner, Josiah; Antoniou, Chloe; Sosnick, Tobin R; Weiss, Eric L; Glotzer, Michael

2012-03-04

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Towards fully automated structure-based function prediction in structural genomics: a case study.

PubMed

Watson, James D; Sanderson, Steve; Ezersky, Alexandra; Savchenko, Alexei; Edwards, Aled; Orengo, Christine; Joachimiak, Andrzej; Laskowski, Roman A; Thornton, Janet M

2007-04-13

As the global Structural Genomics projects have picked up pace, the number of structures annotated in the Protein Data Bank as hypothetical protein or unknown function has grown significantly. A major challenge now involves the development of computational methods to assign functions to these proteins accurately and automatically. As part of the Midwest Center for Structural Genomics (MCSG) we have developed a fully automated functional analysis server, ProFunc, which performs a battery of analyses on a submitted structure. The analyses combine a number of sequence-based and structure-based methods to identify functional clues. After the first stage of the Protein Structure Initiative (PSI), we review the success of the pipeline and the importance of structure-based function prediction. As a dataset, we have chosen all structures solved by the MCSG during the 5 years of the first PSI. Our analysis suggests that two of the structure-based methods are particularly successful and provide examples of local similarity that is difficult to identify using current sequence-based methods. No one method is successful in all cases, so, through the use of a number of complementary sequence and structural approaches, the ProFunc server increases the chances that at least one method will find a significant hit that can help elucidate function. Manual assessment of the results is a time-consuming process and subject to individual interpretation and human error. We present a method based on the Gene Ontology (GO) schema using GO-slims that can allow the automated assessment of hits with a success rate approaching that of expert manual assessment.

Global analysis of ubiquitome in PRRSV-infected pulmonary alveolar macrophages.

PubMed

Zhang, Huan; Fang, Liurong; Zhu, Xinyu; Wang, Dang; Xiao, Shaobo

2018-06-18

Protein lysine ubiquitination is a dynamic reversible post-translational modification that plays key roles in modulating different cellular processes. Porcine reproductive and respiratory syndrome virus (PRRSV) is a notorious pathogen, causing tremendous economic losses for the global swine industry. The possible involvement of ubiquitination in PRRSV infection is unclear. So anti-ubiquitination-based enrichment and LC-MS were performed to investigate the global ubiquitination events triggered by PRRSV infection in pulmonary alveolar macrophages. We totally identified 4044 lysine ubiquitination sites on 1580 cellular proteins, of which 983 sites on 717 proteins were significantly altered at 36 h postinfection. A systematic, intensive bioinformatic analysis of the ubiquitome data suggested that PRRSV suppresses the host immune responses by manipulating the ubiquitination of important adaptors and effectors, including TRAF6, JAK1, STAT1, and ISGs. Ubiquitination was also observed on 15 PRRSV proteins, including important virus proteases and structural proteins that function in virus infectivity and neutralizing antibody elicitation. The efficient replication of PRRSV requires an intact ubiquitin-proteasome system. Our study is the first to analyze the global ubiquitination events in pulmonary alveolar macrophages during PRRSV infection. It provides insight into the molecular mechanisms of PRRSV pathogenesis, promoting the development of antiviral drugs. PRRSV is a notorious pathogen which has been resulting in huge economic losses in the swine industry since the first outbreak. Therefore, more in-depth knowledge of the PRRSV immunoregulatory mechanisms and valid control methods to combat the virus are urgently needed. Ubiquitination is an important post-translational modification regulating various cellular processes. However, information about the possible involvement of ubiquitination responses to PRRSV infection is limited. In this study, a quantitative proteomic approach was first used to analyze ubiquitination level alteration in PRRSV-infected PAMs. We demonstrate that PRRSV can suppresses the host immune responses by manipulating the ubiquitination of important effectors that include TRAF6, JAK1, STAT1, and ISGs. Furthermore, 15 PRRSV proteins undergo ubiquitination and efficient replication of PRRSV requires an intact ubiquitin-proteasome system. Our study will significantly expand our knowledge about the molecular mechanisms of PRRSV pathogenesis and provides novel insights into the development of antiviral drugs. Copyright © 2018. Published by Elsevier B.V.

Integrated Left Ventricular Global Transcriptome and Proteome Profiling in Human End-Stage Dilated Cardiomyopathy.

PubMed

Colak, Dilek; Alaiya, Ayodele A; Kaya, Namik; Muiya, Nzioka P; AlHarazi, Olfat; Shinwari, Zakia; Andres, Editha; Dzimiri, Nduna

2016-01-01

The disease pathways leading to idiopathic dilated cardiomyopathy (DCM) are still elusive. The present study investigated integrated global transcriptional and translational changes in human DCM for disease biomarker discovery. We used identical myocardial tissues from five DCM hearts compared to five non-failing (NF) donor hearts for both transcriptome profiling using the ABI high-density oligonucleotide microarrays and proteome expression with One-Dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on the Synapt G2 system. We identified 1262 differentially expressed genes (DEGs) and 269 proteins (DEPs) between DCM cases and healthy controls. Among the most significantly upregulated (>5-fold) proteins were GRK5, APOA2, IGHG3, ANXA6, HSP90AA1, and ATP5C1 (p< 0.01). On the other hand, the most significantly downregulated proteins were GSTM5, COX17, CAV1 and ANXA3. At least ten entities were concomitantly upregulated on the two analysis platforms: GOT1, ALDH4A1, PDHB, BDH1, SLC2A11, HSP90AA1, HSP90AB1, H2AFV, HSPA5 and NDUFV1. Gene ontology analyses of DEGs and DEPs revealed significant overlap with enrichment of genes/proteins related to metabolic process, biosynthetic process, cellular component organization, oxidative phosphorylation, alterations in glycolysis and ATP synthesis, Alzheimer's disease, chemokine-mediated inflammation and cytokine signalling pathways. The concomitant use of transcriptome and proteome expression to evaluate global changes in DCM has led to the identification of sixteen commonly altered entities as well as novel genes, proteins and pathways whose cardiac functions have yet to be deciphered. This data should contribute towards better management of the disease.

Proteomic Identification and Analysis of Arginine-Methylated Proteins of Plasmodium falciparum at Asexual Blood Stages.

PubMed

Zeeshan, Mohammad; Kaur, Inderjeet; Joy, Joseph; Saini, Ekta; Paul, Gourab; Kaushik, Abhinav; Dabral, Surbhi; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

2017-02-03

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR, or WxxxR. We identified Plasmodium homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms. PMID:28095400

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms.

Plant Comparative and Functional Genomics

DOE PAGES

Yang, Xiaohan; Leebens-Mack, Jim; Chen, Feng; ...

2015-01-01

Plants form the foundation for our global ecosystem and are essential for environmental and human health. An increasing number of available plant genomes and tractable experimental systems, comparative and functional plant genomics research is greatly expanding our knowledge of the molecular basis of economically and nutritionally important traits in crop plants. Inferences drawn from comparative genomics are motivating experimental investigations of gene function and gene interactions. In this special issue aims to highlight recent advances made in comparative and functional genomics research in plants. Nine original research articles in this special issue cover five important topics: (1) transcription factor genemore » families relevant to abiotic stress tolerance; (2) plant secondary metabolism; (3) transcriptomebased markers for quantitative trait locus; (4) epigenetic modifications in plant-microbe interactions; and (5) computational prediction of protein-protein interactions. Finally, we studied the plant species in these articles which include model species as well as nonmodel plant species of economic importance (e.g., food crops and medicinal plants).« less

Plant Comparative and Functional Genomics

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

Yang, Xiaohan; Leebens-Mack, Jim; Chen, Feng

Plants form the foundation for our global ecosystem and are essential for environmental and human health. An increasing number of available plant genomes and tractable experimental systems, comparative and functional plant genomics research is greatly expanding our knowledge of the molecular basis of economically and nutritionally important traits in crop plants. Inferences drawn from comparative genomics are motivating experimental investigations of gene function and gene interactions. In this special issue aims to highlight recent advances made in comparative and functional genomics research in plants. Nine original research articles in this special issue cover five important topics: (1) transcription factor genemore » families relevant to abiotic stress tolerance; (2) plant secondary metabolism; (3) transcriptomebased markers for quantitative trait locus; (4) epigenetic modifications in plant-microbe interactions; and (5) computational prediction of protein-protein interactions. Finally, we studied the plant species in these articles which include model species as well as nonmodel plant species of economic importance (e.g., food crops and medicinal plants).« less

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

PubMed Central

Girard, Eric; Marchal, Stéphane; Perez, Javier; Finet, Stéphanie; Kahn, Richard; Fourme, Roger; Marassio, Guillaume; Dhaussy, Anne-Claire; Prangé, Thierry; Giffard, Marion; Dulin, Fabienne; Bonneté, Françoise; Lange, Reinhard; Abraini, Jacques H.; Mezouar, Mohamed; Colloc'h, Nathalie

2010-01-01

Abstract Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms. PMID:20483346

Myocardial oxidative metabolism and protein synthesis during mechanical circulatory support by extracorporeal membrane oxygenation.

PubMed

Priddy, Colleen M O'Kelly; Kajimoto, Masaki; Ledee, Dolena R; Bouchard, Bertrand; Isern, Nancy; Olson, Aaron K; Des Rosiers, Christine; Portman, Michael A

2013-02-01

Extracorporeal membrane oxygenation (ECMO) provides essential mechanical circulatory support necessary for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur, which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative metabolism and protein synthesis. We focused on the amino acid leucine and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart 1) the fractional contribution of leucine (FcLeucine) and pyruvate to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and 2) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 h of normal circulation or ECMO) and intracoronary infusion [(13)C(6),(15)N]-L-leucine (3.7 mM) alone or with [2-(13)C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (∼40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining 1) metabolic flexibility indicated by ability to respond to pyruvate and 2) a normal or increased capacity for global protein synthesis.

ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?

PubMed Central

Buscà, Roser; Pouysségur, Jacques; Lenormand, Philippe

2016-01-01

The MAP kinase signaling cascade Ras/Raf/MEK/ERK has been involved in a large variety of cellular and physiological processes that are crucial for life. Many pathological situations have been associated to this pathway. More than one isoform has been described at each level of the cascade. In this review we devoted our attention to ERK1 and ERK2, which are the effector kinases of the pathway. Whether ERK1 and ERK2 specify functional differences or are in contrast functionally redundant, constitutes an ongoing debate despite the huge amount of studies performed to date. In this review we compiled data on ERK1 vs. ERK2 gene structures, protein sequences, expression levels, structural and molecular mechanisms of activation and substrate recognition. We have also attempted to perform a rigorous analysis of studies regarding the individual roles of ERK1 and ERK2 by the means of morpholinos, siRNA, and shRNA silencing as well as gene disruption or gene replacement in mice. Finally, we comment on a recent study of gene and protein evolution of ERK isoforms as a distinct approach to address the same question. Our review permits the evaluation of the relevance of published studies in the field especially when measurements of global ERK activation are taken into account. Our analysis favors the hypothesis of ERK1 and ERK2 exhibiting functional redundancy and points to the concept of the global ERK quantity, and not isoform specificity, as being the essential determinant to achieve ERK function. PMID:27376062

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

PubMed

Brenman, J E; Gao, F B; Jan, L Y; Jan, Y N

2001-11-01

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

Endoplasmic reticulum proteostasis impairment in aging.

PubMed

Martínez, Gabriela; Duran-Aniotz, Claudia; Cabral-Miranda, Felipe; Vivar, Juan P; Hetz, Claudio

2017-08-01

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one-third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Attenuated XPC Expression Is Not Associated with Impaired DNA Repair in Bladder Cancer

PubMed Central

Naipal, Kishan A. T.; Raams, Anja; Bruens, Serena T.; Brandsma, Inger; Verkaik, Nicole S.; Jaspers, Nicolaas G. J.; Hoeijmakers, Jan H. J.; van Leenders, Geert J. L. H.; Pothof, Joris; Kanaar, Roland; Boormans, Joost; van Gent, Dik C.

2015-01-01

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors. PMID:25927440

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

Wide range of interacting partners of pea Gβ subunit of G-proteins suggests its multiple functions in cell signalling.

PubMed

Bhardwaj, Deepak; Lakhanpaul, Suman; Tuteja, Narendra

2012-09-01

Climate change is a major concern especially in view of the increasing global population and food security. Plant scientists need to look for genetic tools whose appropriate usage can contribute to sustainable food availability. G-proteins have been identified as some of the potential genetic tools that could be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of three subunits Gα, Gβ and Gγ are important components of a number of signalling pathways. Their structure and functions are already well studied in animals but their potential in plants is now gaining attention for their role in stress tolerance. Earlier we have reported that over expressing pea Gβ conferred heat tolerance in tobacco plants. Here we report the interacting partners (proteins) of Gβ subunit of Pisum sativum and their putative role in stress and development. Out of 90 transformants isolated from the yeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence in multiple experiments. These interacting partners were confirmed using β-galactosidase colony filter lift and ONPG (O-nitrophenyl-β-D-galactopyranoside) assays. These partners include thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gβ subunit in stress signal transduction and development pathways owing to its capability to interact with a wide range of proteins of multiple functions. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Patterns of HIV-1 Protein Interaction Identify Perturbed Host-Cellular Subsystems

PubMed Central

MacPherson, Jamie I.; Dickerson, Jonathan E.; Pinney, John W.; Robertson, David L.

2010-01-01

Human immunodeficiency virus type 1 (HIV-1) exploits a diverse array of host cell functions in order to replicate. This is mediated through a network of virus-host interactions. A variety of recent studies have catalogued this information. In particular the HIV-1, Human Protein Interaction Database (HHPID) has provided a unique depth of protein interaction detail. However, as a map of HIV-1 infection, the HHPID is problematic, as it contains curation error and redundancy; in addition, it is based on a heterogeneous set of experimental methods. Based on identifying shared patterns of HIV-host interaction, we have developed a novel methodology to delimit the core set of host-cellular functions and their associated perturbation from the HHPID. Initially, using biclustering, we identify 279 significant sets of host proteins that undergo the same types of interaction. The functional cohesiveness of these protein sets was validated using a human protein-protein interaction network, gene ontology annotation and sequence similarity. Next, using a distance measure, we group host protein sets and identify 37 distinct higher-level subsystems. We further demonstrate the biological significance of these subsystems by cross-referencing with global siRNA screens that have been used to detect host factors necessary for HIV-1 replication, and investigate the seemingly small intersect between these data sets. Our results highlight significant host-cell subsystems that are perturbed during the course of HIV-1 infection. Moreover, we characterise the patterns of interaction that contribute to these perturbations. Thus, our work disentangles the complex set of HIV-1-host protein interactions in the HHPID, reconciles these with siRNA screens and provides an accessible and interpretable map of infection. PMID:20686668

The use of experimental structures to model protein dynamics.

PubMed

Katebi, Ataur R; Sankar, Kannan; Jia, Kejue; Jernigan, Robert L

2015-01-01

The number of solved protein structures submitted in the Protein Data Bank (PDB) has increased dramatically in recent years. For some specific proteins, this number is very high-for example, there are over 550 solved structures for HIV-1 protease, one protein that is essential for the life cycle of human immunodeficiency virus (HIV) which causes acquired immunodeficiency syndrome (AIDS) in humans. The large number of structures for the same protein and its variants include a sample of different conformational states of the protein. A rich set of structures solved experimentally for the same protein has information buried within the dataset that can explain the functional dynamics and structural mechanism of the protein. To extract the dynamics information and functional mechanism from the experimental structures, this chapter focuses on two methods-Principal Component Analysis (PCA) and Elastic Network Models (ENM). PCA is a widely used statistical dimensionality reduction technique to classify and visualize high-dimensional data. On the other hand, ENMs are well-established simple biophysical method for modeling the functionally important global motions of proteins. This chapter covers the basics of these two. Moreover, an improved ENM version that utilizes the variations found within a given set of structures for a protein is described. As a practical example, we have extracted the functional dynamics and mechanism of HIV-1 protease dimeric structure by using a set of 329 PDB structures of this protein. We have described, step by step, how to select a set of protein structures, how to extract the needed information from the PDB files for PCA, how to extract the dynamics information using PCA, how to calculate ENM modes, how to measure the congruency between the dynamics computed from the principal components (PCs) and the ENM modes, and how to compute entropies using the PCs. We provide the computer programs or references to software tools to accomplish each step and show how to use these programs and tools. We also include computer programs to generate movies based on PCs and ENM modes and describe how to visualize them.

The Use of Experimental Structures to Model Protein Dynamics

PubMed Central

Katebi, Ataur R.; Sankar, Kannan; Jia, Kejue; Jernigan, Robert L.

2014-01-01

Summary The number of solved protein structures submitted in the Protein Data Bank (PDB) has increased dramatically in recent years. For some specific proteins, this number is very high – for example, there are over 550 solved structures for HIV-1 protease, one protein that is essential for the life cycle of human immunodeficiency virus (HIV) which causes acquired immunodeficiency syndrome (AIDS) in humans. The large number of structures for the same protein and its variants include a sample of different conformational states of the protein. A rich set of structures solved experimentally for the same protein has information buried within the dataset that can explain the functional dynamics and structural mechanism of the protein. To extract the dynamics information and functional mechanism from the experimental structures, this chapter focuses on two methods – Principal Component Analysis (PCA) and Elastic Network Models (ENM). PCA is a widely used statistical dimensionality reduction technique to classify and visualize high-dimensional data. On the other hand, ENMs are well-established simple biophysical method for modeling the functionally important global motions of proteins. This chapter covers the basics of these two. Moreover, an improved ENM version that utilizes the variations found within a given set of structures for a protein is described. As a practical example, we have extracted the functional dynamics and mechanism of HIV-1 protease dimeric structure by using a set of 329 PDB structures of this protein. We have described, step by step, how to select a set of protein structures, how to extract the needed information from the PDB files for PCA, how to extract the dynamics information using PCA, how to calculate ENM modes, how to measure the congruency between the dynamics computed from the principal components (PCs) and the ENM modes, and how to compute entropies using the PCs. We provide the computer programs or references to software tools to accomplish each step and show how to use these programs and tools. We also include computer programs to generate movies based on PCs and ENM modes and describe how to visualize them. PMID:25330965

Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

PubMed Central

Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

2014-01-01

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

Protein structure modeling for CASP10 by multiple layers of global optimization.

PubMed

Joo, Keehyoung; Lee, Juyong; Sim, Sangjin; Lee, Sun Young; Lee, Kiho; Heo, Seungryong; Lee, In-Ho; Lee, Sung Jong; Lee, Jooyoung

2014-02-01

In the template-based modeling (TBM) category of CASP10 experiment, we introduced a new protocol called protein modeling system (PMS) to generate accurate protein structures in terms of side-chains as well as backbone trace. In the new protocol, a global optimization algorithm, called conformational space annealing (CSA), is applied to the three layers of TBM procedure: multiple sequence-structure alignment, 3D chain building, and side-chain re-modeling. For 3D chain building, we developed a new energy function which includes new distance restraint terms of Lorentzian type (derived from multiple templates), and new energy terms that combine (physical) energy terms such as dynamic fragment assembly (DFA) energy, DFIRE statistical potential energy, hydrogen bonding term, etc. These physical energy terms are expected to guide the structure modeling especially for loop regions where no template structures are available. In addition, we developed a new quality assessment method based on random forest machine learning algorithm to screen templates, multiple alignments, and final models. For TBM targets of CASP10, we find that, due to the combination of three stages of CSA global optimizations and quality assessment, the modeling accuracy of PMS improves at each additional stage of the protocol. It is especially noteworthy that the side-chains of the final PMS models are far more accurate than the models in the intermediate steps. Copyright © 2013 Wiley Periodicals, Inc.

The Mediator complex: a central integrator of transcription

PubMed Central

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Polycyclic aromatic hydrocarbon metabolic network in Mycobacterium vanbaalenii PYR-1.

PubMed

Kweon, Ohgew; Kim, Seong-Jae; Holland, Ricky D; Chen, Hongyan; Kim, Dae-Wi; Gao, Yuan; Yu, Li-Rong; Baek, Songjoon; Baek, Dong-Heon; Ahn, Hongsik; Cerniglia, Carl E

2011-09-01

This study investigated a metabolic network (MN) from Mycobacterium vanbaalenii PYR-1 for polycyclic aromatic hydrocarbons (PAHs) from the perspective of structure, behavior, and evolution, in which multilayer omics data are integrated. Initially, we utilized a high-throughput proteomic analysis to assess the protein expression response of M. vanbaalenii PYR-1 to seven different aromatic compounds. A total of 3,431 proteins (57.38% of the genome-predicted proteins) were identified, which included 160 proteins that seemed to be involved in the degradation of aromatic hydrocarbons. Based on the proteomic data and the previous metabolic, biochemical, physiological, and genomic information, we reconstructed an experiment-based system-level PAH-MN. The structure of PAH-MN, with 183 metabolic compounds and 224 chemical reactions, has a typical scale-free nature. The behavior and evolution of the PAH-MN reveals a hierarchical modularity with funnel effects in structure/function and intimate association with evolutionary modules of the functional modules, which are the ring cleavage process (RCP), side chain process (SCP), and central aromatic process (CAP). The 189 commonly upregulated proteins in all aromatic hydrocarbon treatments provide insights into the global adaptation to facilitate the PAH metabolism. Taken together, the findings of our study provide the hierarchical viewpoint from genes/proteins/metabolites to the network via functional modules of the PAH-MN equipped with the engineering-driven approaches of modularization and rationalization, which may expand our understanding of the metabolic potential of M. vanbaalenii PYR-1 for bioremediation applications.

Soft Computing Techniques for the Protein Folding Problem on High Performance Computing Architectures.

PubMed

Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M

2016-01-01

The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.

Proteome Characterization of Leaves in Common Bean

PubMed Central

Robison, Faith M.; Heuberger, Adam L.; Brick, Mark A.; Prenni, Jessica E.

2015-01-01

Dry edible bean (Phaseolus vulgaris L.) is a globally relevant food crop. The bean genome was recently sequenced and annotated allowing for proteomics investigations aimed at characterization of leaf phenotypes important to agriculture. The objective of this study was to utilize a shotgun proteomics approach to characterize the leaf proteome and to identify protein abundance differences between two bean lines with known variation in their physiological resistance to biotic stresses. Overall, 640 proteins were confidently identified. Among these are proteins known to be involved in a variety of molecular functions including oxidoreductase activity, binding peroxidase activity, and hydrolase activity. Twenty nine proteins were found to significantly vary in abundance (p-value < 0.05) between the two bean lines, including proteins associated with biotic stress. To our knowledge, this work represents the first large scale shotgun proteomic analysis of beans and our results lay the groundwork for future studies designed to investigate the molecular mechanisms involved in pathogen resistance. PMID:28248269

Fast and Accurate Multivariate Gaussian Modeling of Protein Families: Predicting Residue Contacts and Protein-Interaction Partners

PubMed Central

Feinauer, Christoph; Procaccini, Andrea; Zecchina, Riccardo; Weigt, Martin; Pagnani, Andrea

2014-01-01

In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation) have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids), exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i) the prediction of residue-residue contacts in proteins, and (ii) the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code. PMID:24663061

p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins

PubMed Central

Lee, Sang Hyun; McCormick, Frank

2006-01-01

p97 (also referred to as DAP5, NAT1 or eIF4G2) has been proposed to act as a repressor of protein synthesis. However, we found that p97 is abundantly expressed in proliferating cells and p97 is recruited to ribosomes following growth factor stimulation. We also report that p97 binds eIF2β through its C-terminal domain and localizes to ribosome through its N-terminal MIF4G domain. When overexpressed, p97 increases reporter luciferase activity. In contrast, overexpression of the C-terminal two-thirds of eukaryotic initiation factor 4GI (eIF4GI), a region that shares significant homology with p97, or the N-terminal MIF4G domain of p97 markedly inhibits reporter activity, the rate of global translation and cell proliferation. Conversely, downregulation of p97 levels by RNA interference also decreases the rate of global translation and inhibits cell proliferation. This coincides with an increase in p27/Kip1 protein levels and a marked decrease in CDK2 kinase activity. Taken together, our results demonstrate that p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI and it can positively promote protein synthesis and cell proliferation. PMID:16932749

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

PubMed Central

Byron, Adam; Humphries, Jonathan D.; Randles, Michael J.; Carisey, Alex; Murphy, Stephanie; Knight, David; Brenchley, Paul E.; Zent, Roy; Humphries, Martin J.

2014-01-01

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456. PMID:24436468

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

An Aromatic Sensor with Aversion to Damaged Strands Confers Versatility to DNA Repair

PubMed Central

Maillard, Olivier; Solyom, Szilvia; Naegeli, Hanspeter

2007-01-01

It was not known how xeroderma pigmentosum group C (XPC) protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix. PMID:17355181

Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein

PubMed Central

Wang, Yong; Tang, Chun; Wang, Erkang; Wang, Jin

2012-01-01

An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional “backtracking” behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model. PMID:22532792

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

Thermosensitivity of growth is determined by chaperone-mediated proteome reallocation

PubMed Central

Chen, Ke; Gao, Ye; Mih, Nathan; O’Brien, Edward J.; Yang, Laurence; Palsson, Bernhard O.

2017-01-01

Maintenance of a properly folded proteome is critical for bacterial survival at notably different growth temperatures. Understanding the molecular basis of thermoadaptation has progressed in two main directions, the sequence and structural basis of protein thermostability and the mechanistic principles of protein quality control assisted by chaperones. Yet we do not fully understand how structural integrity of the entire proteome is maintained under stress and how it affects cellular fitness. To address this challenge, we reconstruct a genome-scale protein-folding network for Escherichia coli and formulate a computational model, FoldME, that provides statistical descriptions of multiscale cellular response consistent with many datasets. FoldME simulations show (i) that the chaperones act as a system when they respond to unfolding stress rather than achieving efficient folding of any single component of the proteome, (ii) how the proteome is globally balanced between chaperones for folding and the complex machinery synthesizing the proteins in response to perturbation, (iii) how this balancing determines growth rate dependence on temperature and is achieved through nonspecific regulation, and (iv) how thermal instability of the individual protein affects the overall functional state of the proteome. Overall, these results expand our view of cellular regulation, from targeted specific control mechanisms to global regulation through a web of nonspecific competing interactions that modulate the optimal reallocation of cellular resources. The methodology developed in this study enables genome-scale integration of environment-dependent protein properties and a proteome-wide study of cellular stress responses. PMID:29073085

Transcriptional consequences of XPA disruption in human cell lines

PubMed Central

Manandhar, Mandira; Lowery, Megan G.; Boulware, Karen S.; Lin, Kevin H.; Lu, Yue; Wood, Richard D.

2017-01-01

Nucleotide excision repair (NER) in mammalian cells requires the xeroderma pigmentosum group A protein (XPA) as a core factor. Remarkably, XPA and other NER proteins have been detected by chromatin immunoprecipitation at some active promoters, and NER deficiency is reported to influence the activated transcription of selected genes. However, the global influence of XPA on transcription in human cells has not been determined. We analyzed the human transcriptome by RNA sequencing (RNA-Seq). We first confirmed that XPA is confined to the cell nucleus even in the absence of external DNA damage, in contrast to previous reports that XPA is normally resident in the cytoplasm and is imported following DNA damage. We then analyzed four genetically matched human cell line pairs deficient or proficient in XPA. Of the ∼14,000 genes transcribed in each cell line, 325 genes (2%) had a significant XPA-dependent directional change in gene expression that was common to all four pairs (with a false discovery rate of 0.05). These genes were enriched in pathways for the maintenance of mitochondria. Only 27 common genes were different by more than 1.5-fold. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism. AKR1C2 protein was lower in all of the immortalized XPA-deficient cells. Retinoic acid treatment led to modest XPA-dependent activation of some genes with transcription-related functions. We conclude that XPA status does not globally influence human gene transcription. However, XPA significantly influences expression of a small subset of genes important for mitochondrial functions and steroid hormone metabolism. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum. PMID:28704716

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

PubMed Central

2014-01-01

Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

PubMed

Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2014-01-07

Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU

PubMed Central

Berger, Michael; Farcas, Anca; Geertz, Marcel; Zhelyazkova, Petya; Brix, Klaudia; Travers, Andrew; Muskhelishvili, Georgi

2010-01-01

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci—spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid. PMID:20010798

The structure of the S-layer of Clostridium difficile.

PubMed

Bradshaw, William J; Roberts, April K; Shone, Clifford C; Acharya, K Ravi

2018-03-01

The nosocomially acquired pathogen Clostridium difficile is the primary causative agent of antibiotic associated diarrhoea and causes tens of thousands of deaths globally each year. C. difficile presents a paracrystalline protein array on the surface of the cell known as an S-layer. S-layers have been demonstrated to possess a wide range of important functions, which, combined with their inherent accessibility, makes them a promising drug target. The unusually complex S-layer of C. difficile is primarily comprised of the high- and low- molecular weight S-layer proteins, HMW SLP and LMW SLP, formed from the cleavage of the S-layer precursor protein, SlpA, but may also contain up to 28 SlpA paralogues. A model of how the S-layer functions as a whole is required if it is to be exploited in fighting the bacterium. Here, we provide a summary of what is known about the S-layer of C. difficile and each of the paralogues and, considering some of the domains present, suggest potential roles for them.

Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism

PubMed Central

Gorini, Giorgio; Adron Harris, R; Dayne Mayfield, R

2014-01-01

Recent studies have shown that gene regulation is far more complex than previously believed and does not completely explain changes at the protein level. Therefore, the direct study of the proteome, considerably different in both complexity and dynamicity to the genome/transcriptome, has provided unique insights to an increasing number of researchers. During the past decade, extraordinary advances in proteomic techniques have changed the way we can analyze the composition, regulation, and function of protein complexes and pathways underlying altered neurobiological conditions. When combined with complementary approaches, these advances provide the contextual information for decoding large data sets into meaningful biologically adaptive processes. Neuroproteomics offers potential breakthroughs in the field of alcohol research by leading to a deeper understanding of how alcohol globally affects protein structure, function, interactions, and networks. The wealth of information gained from these advances can help pinpoint relevant biomarkers for early diagnosis and improved prognosis of alcoholism and identify future pharmacological targets for the treatment of this addiction. PMID:23900301

Structure and function of Zika virus NS5 protein: perspectives for drug design.

PubMed

Wang, Boxiao; Thurmond, Stephanie; Hai, Rong; Song, Jikui

2018-05-01

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure-function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed.

Cellular functions of the microprocessor.

PubMed

Macias, Sara; Cordiner, Ross A; Cáceres, Javier F

2013-08-01

The microprocessor is a complex comprising the RNase III enzyme Drosha and the double-stranded RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8 gene) that catalyses the nuclear step of miRNA (microRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as an endonuclease. Recent global analyses of microprocessor and Dicer proteins have suggested novel functions for these components independent of their role in miRNA biogenesis. A HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) experiment designed to identify novel substrates of the microprocessor revealed that this complex binds and regulates a large variety of cellular RNAs. The microprocessor-mediated cleavage of several classes of RNAs not only regulates transcript levels, but also modulates alternative splicing events, independently of miRNA function. Importantly, DGCR8 can also associate with other nucleases, suggesting the existence of alternative DGCR8 complexes that may regulate the fate of a subset of cellular RNAs. The aim of the present review is to provide an overview of the diverse functional roles of the microprocessor.

The functional interactome landscape of the human histone deacetylase family

PubMed Central

Joshi, Preeti; Greco, Todd M; Guise, Amanda J; Luo, Yang; Yu, Fang; Nesvizhskii, Alexey I; Cristea, Ileana M

2013-01-01

Histone deacetylases (HDACs) are a diverse family of essential transcriptional regulatory enzymes, that function through the spatial and temporal recruitment of protein complexes. As the composition and regulation of HDAC complexes are only partially characterized, we built the first global protein interaction network for all 11 human HDACs in T cells. Integrating fluorescence microscopy, immunoaffinity purifications, quantitative mass spectrometry, and bioinformatics, we identified over 200 unreported interactions for both well-characterized and lesser-studied HDACs, a subset of which were validated by orthogonal approaches. We establish HDAC11 as a member of the survival of motor neuron complex and pinpoint a functional role in mRNA splicing. We designed a complementary label-free and metabolic-labeling mass spectrometry-based proteomics strategy for profiling interaction stability among different HDAC classes, revealing that HDAC1 interactions within chromatin-remodeling complexes are largely stable, while transcription factors preferentially exist in rapid equilibrium. Overall, this study represents a valuable resource for investigating HDAC functions in health and disease, encompassing emerging themes of HDAC regulation in cell cycle and RNA processing and a deeper functional understanding of HDAC complex stability. PMID:23752268

HPEPDOCK: a web server for blind peptide-protein docking based on a hierarchical algorithm.

PubMed

Zhou, Pei; Jin, Bowen; Li, Hao; Huang, Sheng-You

2018-05-09

Protein-peptide interactions are crucial in many cellular functions. Therefore, determining the structure of protein-peptide complexes is important for understanding the molecular mechanism of related biological processes and developing peptide drugs. HPEPDOCK is a novel web server for blind protein-peptide docking through a hierarchical algorithm. Instead of running lengthy simulations to refine peptide conformations, HPEPDOCK considers the peptide flexibility through an ensemble of peptide conformations generated by our MODPEP program. For blind global peptide docking, HPEPDOCK obtained a success rate of 33.3% in binding mode prediction on a benchmark of 57 unbound cases when the top 10 models were considered, compared to 21.1% for pepATTRACT server. HPEPDOCK also performed well in docking against homology models and obtained a success rate of 29.8% within top 10 predictions. For local peptide docking, HPEPDOCK achieved a high success rate of 72.6% on a benchmark of 62 unbound cases within top 10 predictions, compared to 45.2% for HADDOCK peptide protocol. Our HPEPDOCK server is computationally efficient and consumed an average of 29.8 mins for a global peptide docking job and 14.2 mins for a local peptide docking job. The HPEPDOCK web server is available at http://huanglab.phys.hust.edu.cn/hpepdock/.

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

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

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Comparative Proteomic Analysis of Liver Steatosis and Fibrosis after Oral Hepatotoxicant Administration in Sprague-Dawley Rats.

PubMed

McDyre, B Claire; AbdulHameed, Mohamed Diwan M; Permenter, Matthew G; Dennis, William E; Baer, Christine E; Koontz, Jason M; Boyle, Molly H; Wallqvist, Anders; Lewis, John A; Ippolito, Danielle L

2018-02-01

The past decade has seen an increase in the development and clinical use of biomarkers associated with histological features of liver disease. Here, we conduct a comparative histological and global proteomics analysis to identify coregulated modules of proteins in the progression of hepatic steatosis or fibrosis. We orally administered the reference chemicals bromobenzene (BB) or 4,4'-methylenedianiline (4,4'-MDA) to male Sprague-Dawley rats for either 1 single administration or 5 consecutive daily doses. Livers were preserved for histopathology and global proteomics assessment. Analysis of liver sections confirmed a dose- and time-dependent increase in frequency and severity of histopathological features indicative of lipid accumulation after BB or fibrosis after 4,4'-MDA. BB administration resulted in a dose-dependent increase in the frequency and severity of inflammation and vacuolation. 4,4'-MDA administration resulted in a dose-dependent increase in the frequency and severity of periportal collagen accumulation and inflammation. Pathway analysis identified a time-dependent enrichment of biological processes associated with steatogenic or fibrogenic initiating events, cellular functions, and toxicological states. Differentially expressed protein modules were consistent with the observed histology, placing physiologically linked protein networks into context of the disease process. This study demonstrates the potential for protein modules to provide mechanistic links between initiating events and histopathological outcomes.

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE PAGES

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.; ...

2015-02-16

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

AIP1-mediated stress signaling in atherosclerosis and arteriosclerosis

PubMed Central

Zhang, Jiqin; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

2016-01-01

AIP1 (encoded by the DAB2IP gene), a signaling scaffolding protein, is abundantly expressed in vascular endothelial cells (EC). While it was initially discovered as an ASK1-interacting protein, AIP1 broadly suppresses inflammatory responses triggered by cytokines and stresses such as TNF, LPS, VEGF and ER stress in EC (therefore AIP1 is an Anti-Inflammatory Protein). Human genome-wide association study (GWAS) has identified DAB2IP gene variants conferring susceptibility to cardiovascular diseases. Consistently, a global or vascular EC-specific deletion of DAB2IP in mice strongly enhances inflammatory responses and exacerbates atherosclerosis and graft arteriosclerosis progression in mouse models. Mechanisms for AIP1 function and regulation associated with human cardiovascular diseases need further investigations. PMID:25732743

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

Uterine deletion of Trp53 compromises antioxidant responses in mouse decidua

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

Burnum, Kristin E.; Hirota, Yasushi; Baker, Erin Shammel

2012-09-01

Preterm birth is a global health issue impacting both mothers and children. However, the etiology of preterm birth is not clearly understood. From our recent finding that premature decidual senescence with terminal differentiation is a cause of preterm birth in mice with uterine Trp53 deletion, encoding p53 protein, led us to explore other potential factors that are related to preterm birth. Utilizing proteomics approaches, here we show that 183 candidate proteins cause significant changes in decidua with Trp53 deletion as compared to normal decidua. Functional categorization of these proteins unveiled new pathways that are influenced by p53. In particular, downregulationmore » of a cluster of antioxidant proteins in p53 deficient decidua suggests that increased oxidative stress could be one cause of preterm birth in mice with uterine deletion of Trp53.« less

Characterization of human pineal gland proteome.

PubMed

Yelamanchi, Soujanya D; Kumar, Manish; Madugundu, Anil K; Gopalakrishnan, Lathika; Dey, Gourav; Chavan, Sandip; Sathe, Gajanan; Mathur, Premendu P; Gowda, Harsha; Mahadevan, Anita; Shankar, Susarla K; Prasad, T S Keshava

2016-11-15

The pineal gland is a neuroendocrine gland located at the center of the brain. It is known to regulate various physiological functions in the body through secretion of the neurohormone melatonin. Comprehensive characterization of the human pineal gland proteome has not been undertaken to date. We employed a high-resolution mass spectrometry-based approach to characterize the proteome of the human pineal gland. A total of 5874 proteins were identified from the human pineal gland in this study. Of these, 5820 proteins were identified from the human pineal gland for the first time. Interestingly, 1136 proteins from the human pineal gland were found to contain a signal peptide domain, which indicates the secretory nature of these proteins. An unbiased global proteomic profile of this biomedically important organ should benefit molecular research to unravel the role of the pineal gland in neuropsychiatric and neurodegenerative diseases.

Trafficking arms: oomycete effectors enter host plant cells.

PubMed

Birch, Paul R J; Rehmany, Anne P; Pritchard, Leighton; Kamoun, Sophien; Beynon, Jim L

2006-01-01

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.

Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins

PubMed Central

Perez, Romel B.; Tischer, Alexander; Auton, Matthew; Whitten, Steven T.

2014-01-01

Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins, mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline and alanine to glycine substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (Rh) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the glycine substitutions decreased polyproline II (PPII) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in Rh were not associated with folding. The experiments showed that changes in local PPII structure cause changes in Rh that are variable and that depend on the intrinsic chain propensities of proline and alanine residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed proline and alanine effects on the structures of intrinsically disordered proteins. PMID:25244701

Subtle balance of tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly.

PubMed

Yeo, Giselle C; Tarakanova, Anna; Baldock, Clair; Wise, Steven G; Buehler, Markus J; Weiss, Anthony S

2016-02-01

The assembly of the tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial β sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule's assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly.

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

PubMed

Vohradsky, Jiri; Janda, Ivan; Grünenfelder, Björn; Berndt, Peter; Röder, Daniel; Langen, Hanno; Weiser, Jaroslav; Jenal, Urs

2003-10-01

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics

PubMed Central

Sawada, Hitoshi; Satoh, Noriyuki

2016-01-01

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs. PMID:27253604

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

PubMed

Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

2017-05-01

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

PubMed Central

Akay, Alper; Craig, Ashley; Lehrbach, Nicolas; Larance, Mark; Pourkarimi, Ehsan; Wright, Jane E.; Lamond, Angus; Miska, Eric; Gartner, Anton

2013-01-01

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways. PMID:24258276

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

PubMed Central

Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

2014-01-01

Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

PubMed

Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

2015-08-01

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

PubMed Central

Azevedo, Jacinthe; Garcia, Damien; Pontier, Dominique; Ohnesorge, Stephanie; Yu, Agnes; Garcia, Shahinez; Braun, Laurence; Bergdoll, Marc; Hakimi, Mohamed Ali; Lagrange, Thierry; Voinnet, Olivier

2010-01-01

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans. PMID:20439431

Ancient thioredoxins evolved to modern-day stability-function requirement by altering native state ensemble.

PubMed

Modi, Tushar; Huihui, Jonathan; Ghosh, Kingshuk; Ozkan, S Banu

2018-06-19

Thioredoxins (THRXs)-small globular proteins that reduce other proteins-are ubiquitous in all forms of life, from Archaea to mammals. Although ancestral thioredoxins share sequential and structural similarity with the modern-day (extant) homologues, they exhibit significantly different functional activity and stability. We investigate this puzzle by comparative studies of their (ancient and modern-day THRXs') native state ensemble, as quantified by the dynamic flexibility index (DFI), a metric for the relative resilience of an amino acid to perturbations in the rest of the protein. Clustering proteins using DFI profiles strongly resemble an alternative classification scheme based on their activity and stability. The DFI profiles of the extant proteins are substantially different around the α3, α4 helices and catalytic regions. Likewise, allosteric coupling of the active site with the rest of the protein is different between ancient and extant THRXs, possibly explaining the decreased catalytic activity at low pH with evolution. At a global level, we note that the population of low-flexibility (called hinges) and high-flexibility sites increases with evolution. The heterogeneity (quantified by the variance) in DFI distribution increases with the decrease in the melting temperature typically associated with the evolution of ancient proteins to their modern-day counterparts.This article is part of a discussion meeting issue 'Allostery and molecular machines'. © 2018 The Author(s).

Accurate Simulation and Detection of Coevolution Signals in Multiple Sequence Alignments

PubMed Central

Ackerman, Sharon H.; Tillier, Elisabeth R.; Gatti, Domenico L.

2012-01-01

Background While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones. Methodology/Principal Findings We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the in silico evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs. Conclusions/Significance Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most effective in each case. PMID:23091608

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

PubMed

Lo, Miranda; Murray, Gerald L; Khoo, Chen Ai; Haake, David A; Zuerner, Richard L; Adler, Ben

2010-11-01

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

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

PubMed

Lewis, Leslie A; Astatke, Mekbib; Umekubo, Peter T; Alvi, Shaheen; Saby, Robert; Afrose, Jehan

2011-10-27

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. 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. 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.

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

Long-term Iron and Phosphorus Co-limitation Fundamentally Restructures Protein Biochemistry of High CO2-adapted Trichodesmium

NASA Astrophysics Data System (ADS)

Hutchins, D. A.; Walworth, N. G.; Fu, F.; Webb, E. A.; Saito, M. A.; Moran, D. M.; McIlvin, M.; Lee, M. D.

2016-02-01

Because the globally-distributed diazotrophic cyanobacterium Trichodesmium is a critical new-nitrogen source to nutrient-deplete marine ecosystems, it is crucial to understand its evolutionary responses to global-change factors as they interact with other important environmental controls such as iron and phosphorus limitation. We grew Trichodesmium under multiple iron and phosphorus (co)-limitation scenarios for 1 year following 7 years of adaptation to both present (380-ppm) and future (750-ppm) CO2 concentrations, and discovered a complex metabolic response specific to Fe/P co-limitation, which includes increased growth rates, whole-cell biochemical restructuring, and cell biomass reduction. The interaction of increasing CO2 with this nutrient co-limited state induced an additional set of comprehensive metabolic shifts away from those seen under present day CO2, characterized by upregulation of a new complement of proteins involved in broad cellular functions, core metabolism, and growth. This restructuring reveals a unique co-limited phenotype under Fe/P "balancing" co-limitation, which fundamentally alters traditional interpretations of interactive nutrient limitations and their subsequent controls on key global biogeochemical processes in both the present and future ocean.

Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed.

PubMed

He, Dongli; Wang, Qiong; Li, Ming; Damaris, Rebecca Njeri; Yi, Xingling; Cheng, Zhongyi; Yang, Pingfang

2016-03-04

Regulation of rice seed germination has been shown to mainly occur at post-transcriptional levels, of which the changes on proteome status is a major one. Lysine acetylation and succinylation are two prevalent protein post-translational modifications (PTMs) involved in multiple biological processes, especially for metabolism regulation. To investigate the potential mechanism controlling metabolism regulation in rice seed germination, we performed the lysine acetylation and succinylation analyses simultaneously. Using high-accuracy nano-LC-MS/MS in combination with the enrichment of lysine acetylated or succinylated peptides from digested embryonic proteins of 24 h after imbibition (HAI) rice seed, a total of 699 acetylated sites from 389 proteins and 665 succinylated sites from 261 proteins were identified. Among these modified lysine sites, 133 sites on 78 proteins were commonly modified by two PTMs. The overlapped PTM sites were more likely to be in polar acidic/basic amino acid regions and exposed on the protein surface. Both of the acetylated and succinylated proteins cover nearly all aspects of cellular functions. Ribosome complex and glycolysis/gluconeogenesis-related proteins were significantly enriched in both acetylated and succinylated protein profiles through KEGG enrichment and protein-protein interaction network analyses. The acetyl-CoA and succinyl-CoA metabolism-related enzymes were found to be extensively modified by both modifications, implying the functional interaction between the two PTMs. This study provides a rich resource to examine the modulation of the two PTMs on the metabolism pathway and other biological processes in germinating rice seed.

Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

PubMed Central

König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

2008-01-01

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones. PMID:26619280

Molecular mechanisms of protein aggregation from global fitting of kinetic models.

PubMed

Meisl, Georg; Kirkegaard, Julius B; Arosio, Paolo; Michaels, Thomas C T; Vendruscolo, Michele; Dobson, Christopher M; Linse, Sara; Knowles, Tuomas P J

2016-02-01

The elucidation of the molecular mechanisms by which soluble proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity associated with aggregation reaction networks, the analysis of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quantitative kinetic assays and global fitting, to determine and to verify a molecular mechanism for aggregation reactions that is compatible with experimental kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic experiments that measure the concentration of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic experiment measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-separated text file. We also outline general experimental strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the analysis, we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global analysis of kinetic data without the need for extensive programming or detailed mathematical knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic analysis: determination of constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate constants. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to determining mechanisms, can be completed in <1 d.

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

PubMed Central

Wang, Jigang; Zhang, Jianbin; Lee, Yew-Mun; Koh, Pin-Lang; Ng, Shukie; Bao, Feichao; Lin, Qingsong; Shen, Han-Ming

2016-01-01

ABSTRACT Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQTM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy. PMID:27463841

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy.

PubMed

Wang, Jigang; Zhang, Jianbin; Lee, Yew-Mun; Koh, Pin-Lang; Ng, Shukie; Bao, Feichao; Lin, Qingsong; Shen, Han-Ming

2016-10-02

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ TM ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

FARME DB: a functional antibiotic resistance element database

PubMed Central

Wallace, James C.; Port, Jesse A.; Smith, Marissa N.; Faustman, Elaine M.

2017-01-01

Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates. Database URL: http://staff.washington.edu/jwallace/farme PMID:28077567

Temporal Analysis of the Magnaporthe Oryzae Proteome During Conidial Germination and Cyclic AMP (cAMP)-mediated Appressorium Formation*

PubMed Central

Franck, William L.; Gokce, Emine; Oh, Yeonyee; Muddiman, David C.; Dean, Ralph A.

2013-01-01

Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and highlight biological processes important for pathogenicity. PMID:23665591

Global Genetic Determinants of Mitochondrial DNA Copy Number

PubMed Central

Zhang, Hengshan; Singh, Keshav K.

2014-01-01

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role. PMID:25170845

Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

PubMed Central

2011-01-01

Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo. PMID:21810278

Novel 3D bio-macromolecular bilinear descriptors for protein science: Predicting protein structural classes.

PubMed

Marrero-Ponce, Yovani; Contreras-Torres, Ernesto; García-Jacas, César R; Barigye, Stephen J; Cubillán, Néstor; Alvarado, Ysaías J

2015-06-07

In the present study, we introduce novel 3D protein descriptors based on the bilinear algebraic form in the ℝ(n) space on the coulombic matrix. For the calculation of these descriptors, macromolecular vectors belonging to ℝ(n) space, whose components represent certain amino acid side-chain properties, were used as weighting schemes. Generalization approaches for the calculation of inter-amino acidic residue spatial distances based on Minkowski metrics are proposed. The simple- and double-stochastic schemes were defined as approaches to normalize the coulombic matrix. The local-fragment indices for both amino acid-types and amino acid-groups are presented in order to permit characterizing fragments of interest in proteins. On the other hand, with the objective of taking into account specific interactions among amino acids in global or local indices, geometric and topological cut-offs are defined. To assess the utility of global and local indices a classification model for the prediction of the major four protein structural classes, was built with the Linear Discriminant Analysis (LDA) technique. The developed LDA-model correctly classifies the 92.6% and 92.7% of the proteins on the training and test sets, respectively. The obtained model showed high values of the generalized square correlation coefficient (GC(2)) on both the training and test series. The statistical parameters derived from the internal and external validation procedures demonstrate the robustness, stability and the high predictive power of the proposed model. The performance of the LDA-model demonstrates the capability of the proposed indices not only to codify relevant biochemical information related to the structural classes of proteins, but also to yield suitable interpretability. It is anticipated that the current method will benefit the prediction of other protein attributes or functions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Myocardial Oxidative Metabolism and Protein Synthesis during Mechanical Circulatory Support by Extracorporeal Membrane Oxygenation

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

Priddy, MD, Colleen M.; Kajimoto, Masaki; Ledee, Dolena

2013-02-01

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support essential for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative. We focused on the amino acid leucine, and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart (i) the fractional contribution of leucine (FcLeucine)more » and pyruvate (FCpyruvate) to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and (ii) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 hours of normal circulation or ECMO) and intracoronary infusion [13C6,15N]-L-leucine (3.7 mM) alone or with [2-13C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (~ 40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. Conclusion: The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining (i) metabolic flexibility indicated by ability to respond to pyruvate, and (ii) a normal or increased capacity for global protein synthesis, suggesting an improved protein balance.« less

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins

PubMed Central

Arvola, René M.

2017-01-01

ABSTRACT Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control. PMID:28318367

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins.

PubMed

Arvola, René M; Weidmann, Chase A; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2017-11-02

Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control.

Global analysis of sumoylation function reveals novel insights into development and appressorium-mediated infection of the rice blast fungus.

PubMed

Liu, Caiyun; Li, Zhigang; Xing, Junjie; Yang, Jun; Wang, Zhao; Zhang, Hong; Chen, Deng; Peng, You-Liang; Chen, Xiao-Lin

2018-04-16

Protein post-translational modifications play critical roles in cellular processes, development and stress response. The small ubiquitin-like modifier (SUMO) to proteins is one of the essential modifications in eukaryotes, but its function remains largely unknown in plant pathogenic fungi. We present a comprehensive analysis combined with proteomic, molecular and cellular approaches to explore the roles of sumoylation in the model plant fungal pathogen, Magnaporthe oryzae. We found the SUMO pathway plays key roles in colony growth, conidia formation and virulence to the host, as well as cell-cycle-related phenotypes. Sumoylation is also involved in responding to different stresses. Affinity purification identified 940 putative SUMO substrates, many of which were reported to be involved in development, stress response and infection. Interestingly, four septins were also shown to be sumoylated. Mutation of consensus sumoylation sites in each septin all resulted in reduced virulence to the host and dislocation of septins in appressoria. Moreover, sumoylation is also involved in extracellular secretion of different effector proteins. Our study on the functions of sumoylation provides novel insight into development and infection of the rice blast fungus. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Erythro-megakaryocytic transcription factors associated with hereditary anemia

PubMed Central

Weiss, Mitchell J.

2014-01-01

Most heritable anemias are caused by mutations in genes encoding globins, red blood cell (RBC) membrane proteins, or enzymes in the glycolytic and hexose monophosphate shunt pathways. A less common class of genetic anemia is caused by mutations that alter the functions of erythroid transcription factors (TFs). Many TF mutations associated with heritable anemia cause truncations or amino acid substitutions, resulting in the production of functionally altered proteins. Characterization of these mutant proteins has provided insights into mechanisms of gene expression, hematopoietic development, and human disease. Mutations within promoter or enhancer regions that disrupt TF binding to essential erythroid genes also cause anemia and heritable variations in RBC traits, such as fetal hemoglobin content. Defining the latter may have important clinical implications for de-repressing fetal hemoglobin synthesis to treat sickle cell anemia and β thalassemia. Functionally important alterations in genes encoding TFs or their cognate cis elements are likely to occur more frequently than currently appreciated, a hypothesis that will soon be tested through ongoing genome-wide association studies and the rapidly expanding use of global genome sequencing for human diagnostics. Findings obtained through such studies of RBCs and associated diseases are likely generalizable to many human diseases and quantitative traits. PMID:24652993

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

PubMed

Molloy, Kevin; Shehu, Amarda

2013-01-01

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. 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. 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.

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

DOE PAGES

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; ...

2016-04-28

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

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

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes

PubMed Central

Tiwari, Sandhya P.; Reuter, Nathalie

2016-01-01

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 β-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the β-barrel core is highly rigid, while the α-helices that flank the β-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design. PMID:27015412

A high resolution atlas of gene expression in the domestic sheep (Ovis aries)

PubMed Central

Farquhar, Iseabail L.; Young, Rachel; Lefevre, Lucas; Pridans, Clare; Tsang, Hiu G.; Afrasiabi, Cyrus; Watson, Mick; Whitelaw, C. Bruce; Freeman, Tom C.; Archibald, Alan L.; Hume, David A.

2017-01-01

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of ‘guilt by association’ was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages. PMID:28915238

A high resolution atlas of gene expression in the domestic sheep (Ovis aries).

PubMed

Clark, Emily L; Bush, Stephen J; McCulloch, Mary E B; Farquhar, Iseabail L; Young, Rachel; Lefevre, Lucas; Pridans, Clare; Tsang, Hiu G; Wu, Chunlei; Afrasiabi, Cyrus; Watson, Mick; Whitelaw, C Bruce; Freeman, Tom C; Summers, Kim M; Archibald, Alan L; Hume, David A

2017-09-01

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of 'guilt by association' was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins.

PubMed

Perez, Romel B; Tischer, Alexander; Auton, Matthew; Whitten, Steven T

2014-12-01

Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (R(h)) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PP(II)) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in R(h) were not associated with folding. The experiments showed that changes in local PP(II) structure cause changes in R(h) that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs. © 2014 Wiley Periodicals, Inc.

Pan-genome analysis of human gastric pathogen H. pylori: comparative genomics and pathogenomics approaches to identify regions associated with pathogenicity and prediction of potential core therapeutic targets.

PubMed

Ali, Amjad; Naz, Anam; Soares, Siomar C; Bakhtiar, Marriam; Tiwari, Sandeep; Hassan, Syed S; Hanan, Fazal; Ramos, Rommel; Pereira, Ulisses; Barh, Debmalya; Figueiredo, Henrique César Pereira; Ussery, David W; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2015-01-01

Helicobacter pylori is a human gastric pathogen implicated as the major cause of peptic ulcer and second leading cause of gastric cancer (~70%) around the world. Conversely, an increased resistance to antibiotics and hindrances in the development of vaccines against H. pylori are observed. Pan-genome analyses of the global representative H. pylori isolates consisting of 39 complete genomes are presented in this paper. Phylogenetic analyses have revealed close relationships among geographically diverse strains of H. pylori. The conservation among these genomes was further analyzed by pan-genome approach; the predicted conserved gene families (1,193) constitute ~77% of the average H. pylori genome and 45% of the global gene repertoire of the species. Reverse vaccinology strategies have been adopted to identify and narrow down the potential core-immunogenic candidates. Total of 28 nonhost homolog proteins were characterized as universal therapeutic targets against H. pylori based on their functional annotation and protein-protein interaction. Finally, pathogenomics and genome plasticity analysis revealed 3 highly conserved and 2 highly variable putative pathogenicity islands in all of the H. pylori genomes been analyzed.

Proteomics technique opens new frontiers in mobilome research.

PubMed

Davidson, Andrew D; Matthews, David A; Maringer, Kevin

2017-01-01

A large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the "mobilome," which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the "domestication" of transposon proteins for cellular functions. Although 'omics studies of mobilome genomes and transcriptomes are common, technical challenges have hampered high-throughput global proteomics analyses of transposons. In a recent paper, we overcame these technical hurdles using a technique called "proteomics informed by transcriptomics" (PIT), and thus published the first unbiased global mobilome-derived proteome for any organism (using cell lines derived from the mosquito Aedes aegypti ). In this commentary, we describe our methods in more detail, and summarise our major findings. We also use new genome sequencing data to show that, in many cases, the specific genomic element expressing a given protein can be identified using PIT. This proteomic technique therefore represents an important technological advance that will open new avenues of research into the role that proteins derived from transposons and other repetitive and sequence diverse genetic elements, such as endogenous retroviruses, play in health and disease.

The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation.

PubMed

Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Dotu, Ivan; Martinez-Salas, Encarnación

2018-05-16

Gemin5 is a predominantly cytoplasmic protein that downregulates translation, beyond controlling snRNPs assembly. The C-terminal region harbors a non-canonical RNA-binding site consisting of two domains, RBS1 and RBS2, which differ in RNA-binding capacity and the ability to modulate translation. Here, we show that these domains recognize distinct RNA targets in living cells. Interestingly, the most abundant and exclusive RNA target of the RBS1 domain was Gemin5 mRNA. Biochemical and functional characterization of this target demonstrated that RBS1 polypeptide physically interacts with a predicted thermodynamically stable stem-loop upregulating mRNA translation, thereby counteracting the negative effect of Gemin5 protein on global protein synthesis. In support of this result, destabilization of the stem-loop impairs the stimulatory effect on translation. Moreover, RBS1 stimulates translation of the endogenous Gemin5 mRNA. Hence, although the RBS1 domain downregulates global translation, it positively enhances translation of RNA targets carrying thermodynamically stable secondary structure motifs. This mechanism allows fine-tuning the availability of Gemin5 to play its multiple roles in gene expression control.

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

PubMed Central

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; Price, Morgan N.; Wetmore, Kelly M.; Poole, Farris L.; Deutschbauer, Adam; Arkin, Adam P.

2015-01-01

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library from Pseudomonas stutzeri strain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation. PMID:26452555

Decoding Mechanisms by which Silent Codon Changes Influence Protein Biogenesis and Function

PubMed Central

Bali, Vedrana; Bebok, Zsuzsanna

2015-01-01

Scope Synonymous codon usage has been a focus of investigation since the discovery of the genetic code and its redundancy. The occurrences of synonymous codons vary between species and within genes of the same genome, known as codon usage bias. Today, bioinformatics and experimental data allow us to compose a global view of the mechanisms by which the redundancy of the genetic code contributes to the complexity of biological systems from affecting survival in prokaryotes, to fine tuning the structure and function of proteins in higher eukaryotes. Studies analyzing the consequences of synonymous codon changes in different organisms have revealed that they impact nucleic acid stability, protein levels, structure and function without altering amino acid sequence. As such, synonymous mutations inevitably contribute to the pathogenesis of complex human diseases. Yet, fundamental questions remain unresolved regarding the impact of silent mutations in human disorders. In the present review we describe developments in this area concentrating on mechanisms by which synonymous mutations may affect protein function and human health. Purpose This synopsis illustrates the significance of synonymous mutations in disease pathogenesis. We review the different steps of gene expression affected by silent mutations, and assess the benefits and possible harmful effects of codon optimization applied in the development of therapeutic biologics. Physiological and medical relevance Understanding mechanisms by which synonymous mutations contribute to complex diseases such as cancer, neurodegeneration and genetic disorders, including the limitations of codon-optimized biologics, provides insight concerning interpretation of silent variants and future molecular therapies. PMID:25817479

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

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

PubMed Central

Thiriet, Christophe; Hayes, Jeffrey J.

2009-01-01

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

USDA-ARS?s Scientific Manuscript database

In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

PubMed Central

Gelperin, Daniel M.; White, Michael A.; Wilkinson, Martha L.; Kon, Yoshiko; Kung, Li A.; Wise, Kevin J.; Lopez-Hoyo, Nelson; Jiang, Lixia; Piccirillo, Stacy; Yu, Haiyuan; Gerstein, Mark; Dumont, Mark E.; Phizicky, Eric M.; Snyder, Michael; Grayhack, Elizabeth J.

2005-01-01

Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome. PMID:16322557

Glutamate system, amyloid β peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology

PubMed Central

Revett, Timothy J.; Baker, Glen B.; Jhamandas, Jack; Kar, Satyabrata

2013-01-01

Alzheimer disease is the most prevalent form of dementia globally and is characterized premortem by a gradual memory loss and deterioration of higher cognitive functions and postmortem by neuritic plaques containing amyloid β peptide and neurofibrillary tangles containing phospho-tau protein. Glutamate is the most abundant neurotransmitter in the brain and is essential to memory formation through processes such as long-term potentiation and so might be pivotal to Alzheimer disease progression. This review discusses how the glutamatergic system is impaired in Alzheimer disease and how interactions of amyloid β and glutamate influence synaptic function, tau phosphorylation and neurodegeneration. Interestingly, glutamate not only influences amyloid β production, but also amyloid β can alter the levels of glutamate at the synapse, indicating that small changes in the concentrations of both molecules could influence Alzheimer disease progression. Finally, we describe how the glutamate receptor antagonist, memantine, has been used in the treatment of individuals with Alzheimer disease and discuss its effectiveness. PMID:22894822

Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart.

PubMed

Levitan, Bryana M; Manning, Janet R; Withers, Catherine N; Smith, Jeffrey D; Shaw, Robin M; Andres, Douglas A; Sorrell, Vincent L; Satin, Jonathan

2016-12-01

Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad -/- ) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad -/- confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad -/- . Rad -/- basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support.

Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

DOE PAGES

Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; ...

2015-09-25

Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosicmore » bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.« less

RNA 3D Structural Motifs: Definition, Identification, Annotation, and Database Searching

NASA Astrophysics Data System (ADS)

Nasalean, Lorena; Stombaugh, Jesse; Zirbel, Craig L.; Leontis, Neocles B.

Structured RNA molecules resemble proteins in the hierarchical organization of their global structures, folding and broad range of functions. Structured RNAs are composed of recurrent modular motifs that play specific functional roles. Some motifs direct the folding of the RNA or stabilize the folded structure through tertiary interactions. Others bind ligands or proteins or catalyze chemical reactions. Therefore, it is desirable, starting from the RNA sequence, to be able to predict the locations of recurrent motifs in RNA molecules. Conversely, the potential occurrence of one or more known 3D RNA motifs may indicate that a genomic sequence codes for a structured RNA molecule. To identify known RNA structural motifs in new RNA sequences, precise structure-based definitions are needed that specify the core nucleotides of each motif and their conserved interactions. By comparing instances of each recurrent motif and applying base pair isosteriCity relations, one can identify neutral mutations that preserve its structure and function in the contexts in which it occurs.

Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart

PubMed Central

Levitan, Bryana M.; Manning, Janet R.; Withers, Catherine N.; Smith, Jeffrey D.; Shaw, Robin M.; Andres, Douglas A.; Sorrell, Vincent L.

2016-01-01

Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad−/−) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad−/− confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad−/−. Rad−/− basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support. PMID:27798760

Terahertz Absorption and Circular Dichroism Spectroscopy of Solvated Biopolymers

NASA Astrophysics Data System (ADS)

Xu, Jing; Plaxco, Kevin; Allen, S. James

2006-03-01

Biopolymers are expected to exhibit broad spectral features in the terahertz frequency range, corresponding to their functionally relevant, global and sub-global collective vibrational modes with ˜ picosecond timescale. Recent advances in terahertz technology have stimulated researchers to employ terahertz absorption spectroscopy to directly probe these postulated collective modes. However, these pioneering studies have been limited to dry and, at best, moist samples. Successful isolation of low frequency vibrational activities of solvated biopolymers in their natural water environment has remained elusive, due to the overwhelming attenuation of the terahertz radiation by water. Here we have developed a terahertz absorption and circular dichroism spectrometer suitable for studying biopolymers in biologically relevant water solutions. We have precisely isolated, for the first time, the terahertz absorption of solvated prototypical proteins, Bovine Serum Albumin and Lysozyme, and made important direct comparison to the existing molecular dynamic simulations and normal mode calculations. We have also successfully demonstrated the magnetic circular dichroism in semiconductors, and placed upper bounds on the terahertz circular dichroism signatures of prototypical proteins in water solution.

Proteomics of eukaryotic microorganisms: The medically and biotechnologically important fungal genus Aspergillus.

PubMed

Kniemeyer, Olaf

2011-08-01

Fungal species of the genus Aspergillus play significant roles as model organisms in basic research, as "cell factories" for the production of organic acids, pharmaceuticals or industrially important enzymes and as pathogens causing superficial and invasive infections in animals and humans. The release of the genome sequences of several Aspergillus sp. has paved the way for global analyses of protein expression in Aspergilli including the characterisation of proteins, which have not designated any function. With the application of proteomic methods, particularly 2-D gel and LC-MS/MS-based methods, first insights into the composition of the proteome of Aspergilli under different growth and stress conditions could be gained. Putative targets of global regulators led to the improvement of industrially relevant Aspergillus strains and so far not described Aspergillus antigens have already been discovered. Here, I review the recent proteome data generated for the species Aspergillus nidulans, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Aspergillus flavus and Aspergillus oryzae. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

PubMed Central

Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

2008-01-01

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis

PubMed Central

Kellner, Nikola; Heimel, Kai; Obhof, Theresa; Finkernagel, Florian; Kämper, Jörg

2014-01-01

The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking. PMID:24391515

Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic Pathways.

PubMed

Vranish, James N; Das, Deepika; Barondeau, David P

2016-11-18

Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

PubMed Central

Selle, Kurt; Goh, Yong J.; Johnson, Brant R.; O’Flaherty, Sarah; Andersen, Joakim M.; Barrangou, Rodolphe; Klaenhammer, Todd R.

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall. PMID:28443071

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

PubMed

Selle, Kurt; Goh, Yong J; Johnson, Brant R; O'Flaherty, Sarah; Andersen, Joakim M; Barrangou, Rodolphe; Klaenhammer, Todd R

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus . Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus , suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

The protein histidine phosphatase LHPP is a tumour suppressor.

PubMed

Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

2018-03-29

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

New developments in probing and targeting protein acylation in malaria, leishmaniasis and African sleeping sickness.

PubMed

Ritzefeld, Markus; Wright, Megan H; Tate, Edward W

2018-02-01

Infections by protozoan parasites, such as Plasmodium falciparum or Leishmania donovani, have a significant health, social and economic impact and threaten billions of people living in tropical and sub-tropical regions of developing countries worldwide. The increasing range of parasite strains resistant to frontline therapeutics makes the identification of novel drug targets and the development of corresponding inhibitors vital. Post-translational modifications (PTMs) are important modulators of biology and inhibition of protein lipidation has emerged as a promising therapeutic strategy for treatment of parasitic diseases. In this review we summarize the latest insights into protein lipidation in protozoan parasites. We discuss how recent chemical proteomic approaches have delivered the first global overviews of protein lipidation in these organisms, contributing to our understanding of the role of this PTM in critical metabolic and cellular functions. Additionally, we highlight the development of new small molecule inhibitors to target parasite acyl transferases.

Time, space, and disorder in the expanding proteome universe.

PubMed

Minde, David-Paul; Dunker, A Keith; Lilley, Kathryn S

2017-04-01

Proteins are highly dynamic entities. Their myriad functions require specific structures, but proteins' dynamic nature ranges all the way from the local mobility of their amino acid constituents to mobility within and well beyond single cells. A truly comprehensive view of the dynamic structural proteome includes: (i) alternative sequences, (ii) alternative conformations, (iii) alternative interactions with a range of biomolecules, (iv) cellular localizations, (v) alternative behaviors in different cell types. While these aspects have traditionally been explored one protein at a time, we highlight recently emerging global approaches that accelerate comprehensive insights into these facets of the dynamic nature of protein structure. Computational tools that integrate and expand on multiple orthogonal data types promise to enable the transition from a disjointed list of static snapshots to a structurally explicit understanding of the dynamics of cellular mechanisms. © 2017 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pursuit of proteomic excellence and the excitement in Košice, Slovakia, at the 11th Central and Eastern European Proteomic Conference (CEEPC).

PubMed

Gadher, Suresh; Bhide, Mangesh; Kovarova, Hana

2018-05-01

The Central and Eastern European Proteomic Conference (CEEPC) successfully launched its second decade of proteomics in Košice, Slovakia with a program of systems biology, cellular, clinical, veterinary and sports proteomics. Whilst many conferences are struggling to attract participants, CEEPC with its outstanding track record and unique 'family - feel' packaged with excellent ambiance is thriving and bringing together proteomics experts from academia, industry, scientific specialties, clinics and precision medicine communities interested in resolving mysteries about protein functionalities in health and disease. CEEPC is also renowned for addressing humanitarian global healthcare issues, may it be ageing, chronic diseases or global epidemics. This year CEEPC intertwined with Košice Peace Marathon's pursuit of excellence in sports and initiatives including sports medicine and global peace.

Biodiversity enhances reef fish biomass and resistance to climate change.

PubMed

Duffy, J Emmett; Lefcheck, Jonathan S; Stuart-Smith, Rick D; Navarrete, Sergio A; Edgar, Graham J

2016-05-31

Fishes are the most diverse group of vertebrates, play key functional roles in aquatic ecosystems, and provide protein for a billion people, especially in the developing world. Those functions are compromised by mounting pressures on marine biodiversity and ecosystems. Because of its economic and food value, fish biomass production provides an unusually direct link from biodiversity to critical ecosystem services. We used the Reef Life Survey's global database of 4,556 standardized fish surveys to test the importance of biodiversity to fish production relative to 25 environmental drivers. Temperature, biodiversity, and human influence together explained 47% of the global variation in reef fish biomass among sites. Fish species richness and functional diversity were among the strongest predictors of fish biomass, particularly for the large-bodied species and carnivores preferred by fishers, and these biodiversity effects were robust to potentially confounding influences of sample abundance, scale, and environmental correlations. Warmer temperatures increased biomass directly, presumably by raising metabolism, and indirectly by increasing diversity, whereas temperature variability reduced biomass. Importantly, diversity and climate interact, with biomass of diverse communities less affected by rising and variable temperatures than species-poor communities. Biodiversity thus buffers global fish biomass from climate change, and conservation of marine biodiversity can stabilize fish production in a changing ocean.

Biodiversity enhances reef fish biomass and resistance to climate change

PubMed Central

Duffy, J. Emmett; Lefcheck, Jonathan S.; Navarrete, Sergio A.; Edgar, Graham J.

2016-01-01

Fishes are the most diverse group of vertebrates, play key functional roles in aquatic ecosystems, and provide protein for a billion people, especially in the developing world. Those functions are compromised by mounting pressures on marine biodiversity and ecosystems. Because of its economic and food value, fish biomass production provides an unusually direct link from biodiversity to critical ecosystem services. We used the Reef Life Survey’s global database of 4,556 standardized fish surveys to test the importance of biodiversity to fish production relative to 25 environmental drivers. Temperature, biodiversity, and human influence together explained 47% of the global variation in reef fish biomass among sites. Fish species richness and functional diversity were among the strongest predictors of fish biomass, particularly for the large-bodied species and carnivores preferred by fishers, and these biodiversity effects were robust to potentially confounding influences of sample abundance, scale, and environmental correlations. Warmer temperatures increased biomass directly, presumably by raising metabolism, and indirectly by increasing diversity, whereas temperature variability reduced biomass. Importantly, diversity and climate interact, with biomass of diverse communities less affected by rising and variable temperatures than species-poor communities. Biodiversity thus buffers global fish biomass from climate change, and conservation of marine biodiversity can stabilize fish production in a changing ocean. PMID:27185921

Frustration in protein elastic network models

NASA Astrophysics Data System (ADS)

Lezon, Timothy; Bahar, Ivet

2010-03-01

Elastic network models (ENMs) are widely used for studying the equilibrium dynamics of proteins. The most common approach in ENM analysis is to adopt a uniform force constant or a non-specific distance dependent function to represent the force constant strength. Here we discuss the influence of sequence and structure in determining the effective force constants between residues in ENMs. Using a novel method based on entropy maximization, we optimize the force constants such that they exactly reporduce a subset of experimentally determined pair covariances for a set of proteins. We analyze the optimized force constants in terms of amino acid types, distances, contact order and secondary structure, and we demonstrate that including frustrated interactions in the ENM is essential for accurately reproducing the global modes in the middle of the frequency spectrum.

Sa-Lrp from Sulfolobus acidocaldarius is a versatile, glutamine-responsive, and architectural transcriptional regulator

PubMed Central

Vassart, Amelia; Wolferen, Marleen; Orell, Alvaro; Hong, Ye; Peeters, Eveline; Albers, Sonja-Verena; Charlier, Daniel

2013-01-01

Sa-Lrp is a member of the leucine-responsive regulatory protein (Lrp)-like family of transcriptional regulators in Sulfolobus acidocaldarius. Previously, we demonstrated the binding of Sa-Lrp to the control region of its own gene in vitro. However, the function and cofactor of Sa-Lrp remained an enigma. In this work, we demonstrate that glutamine is the cofactor of Sa-Lrp by inducing the formation of octamers and increasing the DNA-binding affinity and sequence specificity. In vitro protein-DNA interaction assays indicate that Sa-Lrp binds to promoter regions of genes with a variety of functions including ammonia assimilation, transcriptional control, and UV-induced pili synthesis. DNA binding occurs with a specific affinity for AT-rich binding sites, and the protein induces DNA bending and wrapping upon binding, indicating an architectural role of the regulator. Furthermore, by analyzing an Sa-lrp deletion mutant, we demonstrate that the protein affects transcription of some of the genes of which the promoter region is targeted and that it is an important determinant of the cellular aggregation phenotype. Taking all these results into account, we conclude that Sa-Lrp is a glutamine-responsive global transcriptional regulator with an additional architectural role. PMID:23255531

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry.

PubMed

Plemper, Richard K; Brindley, Melinda A; Iorio, Ronald M

2011-06-01

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.

Evolutionary history and functional implications of protein domains and their combinations in eukaryotes.

PubMed

Itoh, Masumi; Nacher, Jose C; Kuma, Kei-ichi; Goto, Susumu; Kanehisa, Minoru

2007-01-01

In higher multicellular eukaryotes, complex protein domain combinations contribute to various cellular functions such as regulation of intercellular or intracellular signaling and interactions. To elucidate the characteristics and evolutionary mechanisms that underlie such domain combinations, it is essential to examine the different types of domains and their combinations among different groups of eukaryotes. We observed a large number of group-specific domain combinations in animals, especially in vertebrates. Examples include animal-specific combinations in tyrosine phosphorylation systems and vertebrate-specific combinations in complement and coagulation cascades. These systems apparently underwent extensive evolution in the ancestors of these groups. In extant animals, especially in vertebrates, animal-specific domains have greater connectivity than do other domains on average, and contribute to the varying number of combinations in each animal subgroup. In other groups, the connectivities of older domains were greater on average. To observe the global behavior of domain combinations during evolution, we traced the changes in domain combinations among animals and fungi in a network analysis. Our results indicate that there is a correlation between the differences in domain combinations among different phylogenetic groups and different global behaviors. Rapid emergence of animal-specific domains was observed in animals, contributing to specific domain combinations and functional diversification, but no such trends were observed in other clades of eukaryotes. We therefore suggest that the strategy for achieving complex multicellular systems in animals differs from that of other eukaryotes.

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

Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp; Ikehata, Hironobu; Mori, Toshio

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cellsmore » was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.« less

Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

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

K Kucera; L Harrison; M Cappello

2011-12-31

Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinantmore » AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.« less

SwissPalm: Protein Palmitoylation database.

PubMed

Blanc, Mathieu; David, Fabrice; Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

SwissPalm: Protein Palmitoylation database

PubMed Central

Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation. PMID:26339475

Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure

PubMed Central

Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan

2016-01-01

Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase. PMID:27934866

Application of a proteomic approach to identify proteins associated with primary graft non-function after liver transplantation.

PubMed

Kornasiewicz, Oskar; Bojarczuk, Kamil; Bugajski, Marek; Golab, Jakub; Krawczyk, Marek

2012-10-01

Primary graft non-function (PNF) is a rare, life-threatening complication of liver transplantation. Increasing use of extended criteria donor pools and high-risk recipients seem to influence the incidence of PNF. Primary failure is associated with high patient morbidity and inferior graft survival. The only available treatment for PNF is emergency hepatic retransplantation, which is also correlated with significant morbidity and mortality. Therefore, researchers are working to identify risk factors of diagnostic value to prevent PNF. The current study attempted to explore liver proteomic patterns in patients with PNF. Using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS), we compared liver protein homogenates from 3 patients with PNF to those obtained from 6 healthy liver samples to identify potential new biomarkers of PNF. Our comparisons revealed 21 proteins with differential expression (13 upregulated and 8 downregulated). Most of these proteins are involved in energy metabolism, lipid metabolism, peptide cleavage, cell differentiation, and apoptosis. Although none of these proteins appeared more than once in separate analyses, this preliminary study shows that two-dimensional gel electrophoresis and LC-MS may allow identification of characteristic proteins to be used as biomarkers of a life-threatening complication of liver transplantation. Larger-scale analyses could improve patient care by finding suitable prognostic and therapeutic options. These data represent the first global proteomic approach to study PNF.

Recent Advances and Applications in Synchrotron X-Ray Protein Footprinting for Protein Structure and Dynamics Elucidation.

PubMed

Gupta, Sayan; Feng, Jun; Chance, Mark; Ralston, Corie

2016-01-01

Synchrotron X-ray Footprinting is a powerful in situ hydroxyl radical labeling method for analysis of protein structure, interactions, folding and conformation change in solution. In this method, water is ionized by high flux density broad band synchrotron X-rays to produce a steady-state concentration of hydroxyl radicals, which then react with solvent accessible side-chains. The resulting stable modification products are analyzed by liquid chromatography coupled to mass spectrometry. A comparative reactivity rate between known and unknown states of a protein provides local as well as global information on structural changes, which is then used to develop structural models for protein function and dynamics. In this review we describe the XF-MS method, its unique capabilities and its recent technical advances at the Advanced Light Source. We provide a comparison of other hydroxyl radical and mass spectrometry based methods with XFMS. We also discuss some of the latest developments in its usage for studying bound water, transmembrane proteins and photosynthetic protein components, and the synergy of the method with other synchrotron based structural biology methods.

A conifer ABI3-interacting protein plays important roles during key transitions of the plant life cycle.

PubMed

Zeng, Ying; Zhao, Tiehan; Kermode, Allison R

2013-01-01

ABI3 (for ABSCISIC ACID INSENSITIVE3), a transcription factor of the abscisic acid signal transduction pathway, plays a major role during seed development, dormancy inception, and dormancy maintenance. This protein appears to also function in meristematic and vegetative plant tissues and under certain stress conditions. We have isolated the ABI3 gene ortholog (CnABI3) from yellow cedar (Callitropsis nootkatensis) and found that it was functionally similar to other ABI3 genes of angiosperms. Here, we report that using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we have identified another protein of yellow cedar (CnAIP2; for CnABI3 INTERACTING PROTEIN2) that physically interacts with CnABI3. Functional analyses revealed that CnAIP2 plays important roles during key transitions in the plant life cycle: (1) CnAIP2 impaired seed development and reduced seed dormancy; (2) CnAIP2 promoted root development, particularly the initiation of lateral roots, and the CnAIP2 gene promoter was exquisitely auxin sensitive; and (3) CnAIP2 promoted the transition from vegetative growth to reproductive initiation (i.e. flowering). The nature of the effects of CnAIP2 on these processes and other evidence place CnAIP2 in the category of a "global" regulator, whose actions are antagonistic to those of ABI3.

Endoplasmic reticulum stress in the pathogenesis of hypertension.

PubMed

Young, Colin N

2017-08-01

What is the topic of this review? This review highlights the emerging role of disruptions in endoplasmic reticulum (ER) function, namely ER stress, as a contributor to hypertension. What advances does it highlight? This review presents an integrative view of ER stress in cardiovascular control systems, including systems within the brain, kidney and peripheral vasculature, as related to development of hypertension. The endoplasmic reticulum (ER) is a cellular organelle specialized in the synthesis, folding, assembly and modification of proteins. In situations of increased protein demand, complex signalling pathways, termed the unfolded protein response, influence a series of cellular feedback loops to control ER function strictly. Although this is initially a compensatory attempt to maintain cellular homeostasis, chronic activation of the unfolded protein response, known as ER stress, leads to sustained changes in cellular function. A growing body of literature points to ER stress in diverse cardioregulatory systems, including the brain, kidney and vasculature, as central to the development of hypertension. Here, these recent findings from essential and obesity-related forms of hypertension are highlighted in an integrative manner, with discussion of the potential upstream causes and downstream consequences of ER stress. Given that hypertension is a leading medical and socio-economic global challenge, emerging findings suggest that targeting ER stress might represent a viable strategy for the treatment of hypertensive disease. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer *

PubMed Central

Hutton, Josiah E.; Wang, Xiaojing; Zimmerman, Lisa J.; Slebos, Robbert J. C.; Trenary, Irina A.; Young, Jamey D.; Li, Ming; Liebler, Daniel C.

2016-01-01

Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25–twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

PubMed

Dinh, Trinh V; Bienvenut, Willy V; Linster, Eric; Feldman-Salit, Anna; Jung, Vincent A; Meinnel, Thierry; Hell, Rüdiger; Giglione, Carmela; Wirtz, Markus

2015-07-01

Protein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947). © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Proteogenomic characterization of human colon and rectal cancer

PubMed Central

Zhang, Bing; Wang, Jing; Wang, Xiaojing; Zhu, Jing; Liu, Qi; Shi, Zhiao; Chambers, Matthew C.; Zimmerman, Lisa J.; Shaddox, Kent F.; Kim, Sangtae; Davies, Sherri R.; Wang, Sean; Wang, Pei; Kinsinger, Christopher R.; Rivers, Robert C.; Rodriguez, Henry; Townsend, R. Reid; Ellis, Matthew J.C.; Carr, Steven A.; Tabb, David L.; Coffey, Robert J.; Slebos, Robbert J.C.; Liebler, Daniel C.

2014-01-01

Summary We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology. PMID:25043054

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

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

Liu, Haijun; Zhang, Hao; King, Jeremy D.

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outsidemore » of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.« less

The impact of protein disulfide bonds on the amyloid fibril morphology

PubMed Central

Kurouski, Dmitry

2014-01-01

Amyloid fibrils are associated with many neurodegenerative diseases. Being formed from more than 20 different proteins that are functionally or structurally unrelated, amyloid fibrils share a common cross-β core structure. It is a well-accepted hypothesis that fibril biological activity and the associated toxicity vary with their morphology. Partial denaturation of a native protein usually precedes the initial stage of fibrillation, namely the nucleation process. Low pH and elevated temperature, typical conditions of amyloid fibril formation in vitro, resulted in partial denaturation of the proteins. Cleavage of disulfide bonds results typically in significant disruption of protein native structure and in the formation of the molten global state. Herein we report on a comparative investigation of fibril formation by apo-α-lactalbumin and its analog that contains only one of the four original disulfide bonds using deep UV resonance and non-resonance Raman spectroscopy and atomic force microscopy. Significant differences in the aggregation mechanism and the resulting fibril morphology were found. PMID:24693331

A Proteomic View at T Cell Costimulation

PubMed Central

Hombach, Andreas A.; Recktenwald, Christian V.; Dressler, Sven P.; Abken, Hinrich; Seliger, Barbara

2012-01-01

The “two-signal paradigm” in T cell activation predicts that the cooperation of “signal 1,” provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with “signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3+ CD69- resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation. PMID:22539942

Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

PubMed Central

Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

2015-01-01

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

Physics-based protein-structure prediction using a hierarchical protocol based on the UNRES force field: assessment in two blind tests.

PubMed

Ołdziej, S; Czaplewski, C; Liwo, A; Chinchio, M; Nanias, M; Vila, J A; Khalili, M; Arnautova, Y A; Jagielska, A; Makowski, M; Schafroth, H D; Kaźmierkiewicz, R; Ripoll, D R; Pillardy, J; Saunders, J A; Kang, Y K; Gibson, K D; Scheraga, H A

2005-05-24

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of alpha and alpha+beta proteins [60-70 residues with C(alpha) rms deviation (rmsd) <6 A]. However, for alpha+beta proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two alpha and three alpha+beta, with sizes of 53-235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue alpha+beta protein from Thermotoga maritima), we predicted the complete, topologically correct structure with 7.3-A C(alpha) rmsd. So far this protein is the largest alpha+beta protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from T. maritima (a 235-residue mainly alpha-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 A rmsd, except the 32 C-terminal residues, most of which form a beta-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.

A random set scoring model for prioritization of disease candidate genes using protein complexes and data-mining of GeneRIF, OMIM and PubMed records.

PubMed

Jiang, Li; Edwards, Stefan M; Thomsen, Bo; Workman, Christopher T; Guldbrandtsen, Bernt; Sørensen, Peter

2014-09-24

Prioritizing genetic variants is a challenge because disease susceptibility loci are often located in genes of unknown function or the relationship with the corresponding phenotype is unclear. A global data-mining exercise on the biomedical literature can establish the phenotypic profile of genes with respect to their connection to disease phenotypes. The importance of protein-protein interaction networks in the genetic heterogeneity of common diseases or complex traits is becoming increasingly recognized. Thus, the development of a network-based approach combined with phenotypic profiling would be useful for disease gene prioritization. We developed a random-set scoring model and implemented it to quantify phenotype relevance in a network-based disease gene-prioritization approach. We validated our approach based on different gene phenotypic profiles, which were generated from PubMed abstracts, OMIM, and GeneRIF records. We also investigated the validity of several vocabulary filters and different likelihood thresholds for predicted protein-protein interactions in terms of their effect on the network-based gene-prioritization approach, which relies on text-mining of the phenotype data. Our method demonstrated good precision and sensitivity compared with those of two alternative complex-based prioritization approaches. We then conducted a global ranking of all human genes according to their relevance to a range of human diseases. The resulting accurate ranking of known causal genes supported the reliability of our approach. Moreover, these data suggest many promising novel candidate genes for human disorders that have a complex mode of inheritance. We have implemented and validated a network-based approach to prioritize genes for human diseases based on their phenotypic profile. We have devised a powerful and transparent tool to identify and rank candidate genes. Our global gene prioritization provides a unique resource for the biological interpretation of data from genome-wide association studies, and will help in the understanding of how the associated genetic variants influence disease or quantitative phenotypes.

Amplitude spectrum distance: measuring the global shape divergence of protein fragments.

PubMed

Galiez, Clovis; Coste, François

2015-08-14

In structural bioinformatics, there is an increasing interest in identifying and understanding the evolution of local protein structures regarded as key structural or functional protein building blocks. A central need is then to compare these, possibly short, fragments by measuring efficiently and accurately their (dis)similarity. Progress towards this goal has given rise to scores enabling to assess the strong similarity of fragments. Yet, there is still a lack of more progressive scores, with meaningful intermediate values, for the comparison, retrieval or clustering of distantly related fragments. We introduce here the Amplitude Spectrum Distance (ASD), a novel way of comparing protein fragments based on the discrete Fourier transform of their C(α) distance matrix. Defined as the distance between their amplitude spectra, ASD can be computed efficiently and provides a parameter-free measure of the global shape dissimilarity of two fragments. ASD inherits from nice theoretical properties, making it tolerant to shifts, insertions, deletions, circular permutations or sequence reversals while satisfying the triangle inequality. The practical interest of ASD with respect to RMSD, RMSDd, BC and TM scores is illustrated through zinc finger retrieval experiments and concrete structure examples. The benefits of ASD are also illustrated by two additional clustering experiments: domain linkers fragments and complementarity-determining regions of antibodies. Taking advantage of the Fourier transform to compare fragments at a global shape level, ASD is an objective and progressive measure taking into account the whole fragments. Its practical computation time and its properties make ASD particularly relevant for applications requiring meaningful measures on distantly related protein fragments, such as similar fragments retrieval asking for high recalls as shown in the experiments, or for any application taking also advantage of triangle inequality, such as fragments clustering. ASD program and source code are freely available at: http://www.irisa.fr/dyliss/public/ASD/.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th‐century pandemics

PubMed Central

Pasricha, Gunisha; Mishra, Akhilesh C.; Chakrabarti, Alok K.

2012-01-01

Please cite this paper as: Pasricha et al. (2012) Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the Influenza A virus subtypes responsible for the 20th‐century pandemics. Influenza and Other Respiratory Viruses 7(4), 497–505. Background  PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Methods  Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Results  Analysis showed that 96·4% of the H5N1 influenza viruses harbored full‐length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th‐century pandemic influenza viruses contained full‐length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human‐ and avian host‐specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Conclusions  Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host‐specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity. PMID:22788742

Randomizing Genome-Scale Metabolic Networks

PubMed Central

Samal, Areejit; Martin, Olivier C.

2011-01-01

Networks coming from protein-protein interactions, transcriptional regulation, signaling, or metabolism may appear to have “unusual” properties. To quantify this, it is appropriate to randomize the network and test the hypothesis that the network is not statistically different from expected in a motivated ensemble. However, when dealing with metabolic networks, the randomization of the network using edge exchange generates fictitious reactions that are biochemically meaningless. Here we provide several natural ensembles of randomized metabolic networks. A first constraint is to use valid biochemical reactions. Further constraints correspond to imposing appropriate functional constraints. We explain how to perform these randomizations with the help of Markov Chain Monte Carlo (MCMC) and show that they allow one to approach the properties of biological metabolic networks. The implication of the present work is that the observed global structural properties of real metabolic networks are likely to be the consequence of simple biochemical and functional constraints. PMID:21779409

Systems Proteomics for Translational Network Medicine

PubMed Central

Arrell, D. Kent; Terzic, Andre

2012-01-01

Universal principles underlying network science, and their ever-increasing applications in biomedicine, underscore the unprecedented capacity of systems biology based strategies to synthesize and resolve massive high throughput generated datasets. Enabling previously unattainable comprehension of biological complexity, systems approaches have accelerated progress in elucidating disease prediction, progression, and outcome. Applied to the spectrum of states spanning health and disease, network proteomics establishes a collation, integration, and prioritization algorithm to guide mapping and decoding of proteome landscapes from large-scale raw data. Providing unparalleled deconvolution of protein lists into global interactomes, integrative systems proteomics enables objective, multi-modal interpretation at molecular, pathway, and network scales, merging individual molecular components, their plurality of interactions, and functional contributions for systems comprehension. As such, network systems approaches are increasingly exploited for objective interpretation of cardiovascular proteomics studies. Here, we highlight network systems proteomic analysis pipelines for integration and biological interpretation through protein cartography, ontological categorization, pathway and functional enrichment and complex network analysis. PMID:22896016

Scattering from Colloid-Polymer Conjugates with Excluded Volume Effect

DOE PAGES

Li, Xin; Sanchez-Diaz, Luis E.; Smith, Gregory Scott; ...

2015-01-13

This work presents scattering functions of conjugates consisting of a colloid particle and a self-avoiding polymer chain as a model for protein-polymer conjugates and nanoparticle-polymer conjugates in solution. The model is directly derived from the two-point correlation function with the inclusion of excluded volume effects. The dependence of the calculated scattering function on the geometric shape of the colloid and polymer stiffness is investigated. The model is able to describe the experimental scattering signature of the solutions of suspending hard particle-polymer conjugates and provide additional conformational information. This model explicitly elucidates the link between the global conformation of a conjugatemore » and the microstructure of its constituent components.« less

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

Independent Metrics for Protein Backbone and Side-Chain Flexibility: Time Scales and Effects of Ligand Binding.

PubMed

Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

2015-03-10

Conformational dynamics are central for understanding biomolecular structure and function, since biological macromolecules are inherently flexible at room temperature and in solution. Computational methods are nowadays capable of providing valuable information on the conformational ensembles of biomolecules. However, analysis tools and intuitive metrics that capture dynamic information from in silico generated structural ensembles are limited. In standard work-flows, flexibility in a conformational ensemble is represented through residue-wise root-mean-square fluctuations or B-factors following a global alignment. Consequently, these approaches relying on global alignments discard valuable information on local dynamics. Results inherently depend on global flexibility, residue size, and connectivity. In this study we present a novel approach for capturing positional fluctuations based on multiple local alignments instead of one single global alignment. The method captures local dynamics within a structural ensemble independent of residue type by splitting individual local and global degrees of freedom of protein backbone and side-chains. Dependence on residue type and size in the side-chains is removed via normalization with the B-factors of the isolated residue. As a test case, we demonstrate its application to a molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) on the millisecond time scale. This allows for illustrating different time scales of backbone and side-chain flexibility. Additionally, we demonstrate the effects of ligand binding on side-chain flexibility of three serine proteases. We expect our new methodology for quantifying local flexibility to be helpful in unraveling local changes in biomolecular dynamics.

Reconstruction of the experimentally supported human protein interactome: what can we learn?

PubMed

Klapa, Maria I; Tsafou, Kalliopi; Theodoridis, Evangelos; Tsakalidis, Athanasios; Moschonas, Nicholas K

2013-10-02

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. 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. 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.

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

PubMed Central

Campos, Rafael K.; Wong, Benjamin; Lu, Yi-Fan; Shi, Pei-Yong; Pompon, Julien

2016-01-01

ABSTRACT The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. IMPORTANCE Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs. PMID:27974556

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

PubMed

Schroeter, Christina B; Koehler, Sybille; Kann, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T; Rinschen, Markus M

2018-04-25

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages.

PubMed

Kaur, Inderjeet; Zeeshan, Mohammad; Saini, Ekta; Kaushik, Abhinav; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

2016-10-20

Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.

Efficient prediction of human protein-protein interactions at a global scale.

PubMed

Schoenrock, Andrew; Samanfar, Bahram; Pitre, Sylvain; Hooshyar, Mohsen; Jin, Ke; Phillips, Charles A; Wang, Hui; Phanse, Sadhna; Omidi, Katayoun; Gui, Yuan; Alamgir, Md; Wong, Alex; Barrenäs, Fredrik; Babu, Mohan; Benson, Mikael; Langston, Michael A; Green, James R; Dehne, Frank; Golshani, Ashkan

2014-12-10

Our knowledge of global protein-protein interaction (PPI) networks in complex organisms such as humans is hindered by technical limitations of current methods. On the basis of short co-occurring polypeptide regions, we developed a tool called MP-PIPE capable of predicting a global human PPI network within 3 months. With a recall of 23% at a precision of 82.1%, we predicted 172,132 putative PPIs. We demonstrate the usefulness of these predictions through a range of experiments. The speed and accuracy associated with MP-PIPE can make this a potential tool to study individual human PPI networks (from genomic sequences alone) for personalized medicine.

The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii.

PubMed

Pérez-Pérez, María Esther; Couso, Inmaculada; Crespo, José L

2017-07-12

Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii . The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae.

The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii

PubMed Central

Pérez-Pérez, María Esther; Crespo, José L.

2017-01-01

Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii. The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae. PMID:28704927

Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats.

PubMed

Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

2017-10-01

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Strategies for the etiological therapy of cystic fibrosis.

PubMed

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-11-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies.

Strategies for the etiological therapy of cystic fibrosis

PubMed Central

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-01-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies. PMID:28937684

A protein interaction network analysis for yeast integral membrane protein.

PubMed

Shi, Ming-Guang; Huang, De-Shuang; Li, Xue-Ling

2008-01-01

Although the yeast Saccharomyces cerevisiae is the best exemplified single-celled eukaryote, the vast number of protein-protein interactions of integral membrane proteins of Saccharomyces cerevisiae have not been characterized by experiments. Here, based on the kernel method of Greedy Kernel Principal Component analysis plus Linear Discriminant Analysis, we identify 300 protein-protein interactions involving 189 membrane proteins and get the outcome of a highly connected protein-protein interactions network. Furthermore, we study the global topological features of integral membrane proteins network of Saccharomyces cerevisiae. These results give the comprehensive description of protein-protein interactions of integral membrane proteins and reveal global topological and robustness of the interactome network at a system level. This work represents an important step towards a comprehensive understanding of yeast protein interactions.

Thiol redox transitions in cell signaling: a lesson from N-acetylcysteine.

PubMed

Parasassi, Tiziana; Brunelli, Roberto; Costa, Graziella; De Spirito, Marco; Krasnowska, Ewa; Lundeberg, Thomas; Pittaluga, Eugenia; Ursini, Fulvio

2010-06-29

The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.

Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

PubMed

Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

2016-03-21

Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant-parasitic nematodes.

A draft annotation and overview of the human genome

PubMed Central

Wright, Fred A; Lemon, William J; Zhao, Wei D; Sears, Russell; Zhuo, Degen; Wang, Jian-Ping; Yang, Hee-Yung; Baer, Troy; Stredney, Don; Spitzner, Joe; Stutz, Al; Krahe, Ralf; Yuan, Bo

2001-01-01

Background The recent draft assembly of the human genome provides a unified basis for describing genomic structure and function. The draft is sufficiently accurate to provide useful annotation, enabling direct observations of previously inferred biological phenomena. Results We report here a functionally annotated human gene index placed directly on the genome. The index is based on the integration of public transcript, protein, and mapping information, supplemented with computational prediction. We describe numerous global features of the genome and examine the relationship of various genetic maps with the assembly. In addition, initial sequence analysis reveals highly ordered chromosomal landscapes associated with paralogous gene clusters and distinct functional compartments. Finally, these annotation data were synthesized to produce observations of gene density and number that accord well with historical estimates. Such a global approach had previously been described only for chromosomes 21 and 22, which together account for 2.2% of the genome. Conclusions We estimate that the genome contains 65,000-75,000 transcriptional units, with exon sequences comprising 4%. The creation of a comprehensive gene index requires the synthesis of all available computational and experimental evidence. PMID:11516338

Embryonic Stem Cell-Based Cardiopatches Improve Cardiac Function in Infarcted Rats

PubMed Central

Vallée, Jean-Paul; Hauwel, Mathieu; Lepetit-Coiffé, Matthieu; Bei, Wang; Montet-Abou, Karin; Meda, Paolo; Gardier, Stephany; Zammaretti, Prisca; Kraehenbuehl, Thomas P.; Herrmann, Francois; Hubbell, Jeffrey A.

2012-01-01

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction. PMID:23197784

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis*

PubMed Central

Wojtuszkiewicz, Anna; Schuurhuis, Gerrit J.; Kessler, Floortje L.; Piersma, Sander R.; Knol, Jaco C.; Pham, Thang V.; Jansen, Gerrit; Musters, René J. P.; van Meerloo, Johan; Assaraf, Yehuda G.; Kaspers, Gertjan J. L.; Zweegman, Sonja; Cloos, Jacqueline; Jimenez, Connie R.

2016-01-01

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance. PMID:26801919

Global effect of the lack of inorganic polyphosphate in the extremophilic archaeon Sulfolobus solfataricus: A proteomic approach.

PubMed

Soto, Daniela F; Recalde, Alejandra; Orell, Alvaro; Albers, Sonja-Verena; Paradela, Alberto; Navarro, Claudio A; Jerez, Carlos A

2018-03-01

Inorganic polyphosphates (polyP) are present in all living cells and several important functions have been described for them. They are involved in the response to stress conditions, such as nutrient depletion, oxidative stress and toxic metals amongst others. A recombinant strain of Sulfolobus solfataricus unable to accumulate polyP was designed by the overexpression of its endogenous ppx gene. The overall impact of the lack of polyP on this S. solfataricus polyP (-) strain was analyzed by using quantitative proteomics (isotope-coded protein label, ICPL). Stress-related proteins, such as peroxiredoxins and heat shock proteins, proteins involved in metabolism and several others were produced at higher levels in the ppx expression strain. The polyP deficient strain showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA gene coding for the P-type copper-exporting ATPase. This implies a complementary function of both copper resistance systems. These results strongly suggests that the lack of polyP makes this hyperthermophilic archaeon more sensitive to toxic conditions, such as an exposure to metals or other harmful stimuli, emphasizing the importance of this inorganic phosphate polymers in the adaptations to live in the environmental conditions in which thermoacidophilic archaea thrive. Inorganic polyphosphate (polyP) are ubiquitous molecules with many functions in living organisms. Few studies related to these polymers have been made in archaea. The construction of a polyP deficient recombinant strain of Sulfolobus solfataricus allowed the study of the global changes in the proteome of this thermoacidophilic archaeon in the absence of polyP compared with the wild type strain. The results obtained using quantitative proteomics suggest an important participation of polyP in the oxidative stress response of the cells and as having a possible metabolic role in the cell, as previously described in bacteria. The polyP deficient strain also showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA, implying a complementary role of both copper resistance systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

PubMed Central

2011-01-01

Background Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29). PMID:22081966

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

PubMed Central

Wang, Qi-En; Zhu, Qianzheng; Wani, Gulzar; El-Mahdy, Mohamed A.; Li, Jinyou; Wani, Altaf A.

2005-01-01

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein. PMID:16030353

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

FACETS: multi-faceted functional decomposition of protein interaction networks

PubMed Central

Seah, Boon-Siew; Bhowmick, Sourav S.; Forbes Dewey, C.

2012-01-01

Motivation: The availability of large-scale curated protein interaction datasets has given rise to the opportunity to investigate higher level organization and modularity within the protein–protein interaction (PPI) network using graph theoretic analysis. Despite the recent progress, systems level analysis of high-throughput PPIs remains a daunting task because of the amount of data they present. In this article, we propose a novel PPI network decomposition algorithm called FACETS in order to make sense of the deluge of interaction data using Gene Ontology (GO) annotations. FACETS finds not just a single functional decomposition of the PPI network, but a multi-faceted atlas of functional decompositions that portray alternative perspectives of the functional landscape of the underlying PPI network. Each facet in the atlas represents a distinct interpretation of how the network can be functionally decomposed and organized. Our algorithm maximizes interpretative value of the atlas by optimizing inter-facet orthogonality and intra-facet cluster modularity. Results: We tested our algorithm on the global networks from IntAct, and compared it with gold standard datasets from MIPS and KEGG. We demonstrated the performance of FACETS. We also performed a case study that illustrates the utility of our approach. Contact: seah0097@ntu.edu.sg or assourav@ntu.edu.sg Supplementary information: Supplementary data are available at the Bioinformatics online. Availability: Our software is available freely for non-commercial purposes from: http://www.cais.ntu.edu.sg/∼assourav/Facets/ PMID:22908217

Identification and analysis of unitary loss of long-established protein-coding genes in Poaceae shows evidences for biased gene loss and putatively functional transcription of relics.

PubMed

Zhao, Yi; Tang, Liang; Li, Zhe; Jin, Jinpu; Luo, Jingchu; Gao, Ge

2015-04-18

Long-established protein-coding genes may lose their coding potential during evolution ("unitary gene loss"). Members of the Poaceae family are a major food source and represent an ideal model clade for plant evolution research. However, the global pattern of unitary gene loss in Poaceae genomes as well as the evolutionary fate of lost genes are still less-investigated and remain largely elusive. Using a locally developed pipeline, we identified 129 unitary gene loss events for long-established protein-coding genes from four representative species of Poaceae, i.e. brachypodium, rice, sorghum and maize. Functional annotation suggested that the lost genes in all or most of Poaceae species are enriched for genes involved in development and response to endogenous stimulus. We also found that 44 mutated genomic loci of lost genes, which we referred as relics, were still actively transcribed, and of which 84% (37 of 44) showed significantly differential expression across different tissues. More interestingly, we found that there were totally five expressed relics may function as competitive endogenous RNA in brachypodium, rice and sorghum genome. Based on comparative genomics and transcriptome data, we firstly compiled a comprehensive catalogue of unitary gene loss events in Poaceae species and characterized a statistically significant functional preference for these lost genes as well showed the potential of relics functioning as competitive endogenous RNAs in Poaceae genomes.