Differential targeting of Gbetagamma-subunit signaling with small molecules.

PubMed

Bonacci, Tabetha M; Mathews, Jennifer L; Yuan, Chujun; Lehmann, David M; Malik, Sundeep; Wu, Dianqing; Font, Jose L; Bidlack, Jean M; Smrcka, Alan V

2006-04-21

G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7.

PubMed

Hussain, Rawaa H; Zawawi, Mariam; Bayfield, Mark A

2013-10-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3'OH-dependent trailer binding/protection and a UUU-3'OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3'OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.

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.

Finding Correlation between Protein Protein Interaction Modules Using Semantic Web Techniques

NASA Astrophysics Data System (ADS)

Kargar, Mehdi; Moaven, Shahrouz; Abolhassani, Hassan

Many complex networks such as social networks and computer show modular structures, where edges between nodes are much denser within modules than between modules. It is strongly believed that cellular networks are also modular, reflecting the relative independence and coherence of different functional units in a cell. In this paper we used a human curated dataset. In this paper we consider each module in the PPI network as ontology. Using techniques in ontology alignment, we compare each pair of modules in the network. We want to see that is there a correlation between the structure of each module or they have totally different structures. Our results show that there is no correlation between proteins in a protein protein interaction network.

Network-Based Disease Module Discovery by a Novel Seed Connector Algorithm with Pathobiological Implications.

PubMed

Wang, Rui-Sheng; Loscalzo, Joseph

2018-05-20

Understanding the genetic basis of complex diseases is challenging. Prior work shows that disease-related proteins do not typically function in isolation. Rather, they often interact with each other to form a network module that underlies dysfunctional mechanistic pathways. Identifying such disease modules will provide insights into a systems-level understanding of molecular mechanisms of diseases. Owing to the incompleteness of our knowledge of disease proteins and limited information on the biological mediators of pathobiological processes, the key proteins (seed proteins) for many diseases appear scattered over the human protein-protein interactome and form a few small branches, rather than coherent network modules. In this paper, we develop a network-based algorithm, called the Seed Connector algorithm (SCA), to pinpoint disease modules by adding as few additional linking proteins (seed connectors) to the seed protein pool as possible. Such seed connectors are hidden disease module elements that are critical for interpreting the functional context of disease proteins. The SCA aims to connect seed disease proteins so that disease mechanisms and pathways can be decoded based on predicted coherent network modules. We validate the algorithm using a large corpus of 70 complex diseases and binding targets of over 200 drugs, and demonstrate the biological relevance of the seed connectors. Lastly, as a specific proof of concept, we apply the SCA to a set of seed proteins for coronary artery disease derived from a meta-analysis of large-scale genome-wide association studies and obtain a coronary artery disease module enriched with important disease-related signaling pathways and drug targets not previously recognized. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

NASA Astrophysics Data System (ADS)

Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

2006-04-01

G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation

PubMed Central

Wu, Chia-Chou; Lin, Che

2015-01-01

The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. PMID:25977693

BinTree Seeking: A Novel Approach to Mine Both Bi-Sparse and Cohesive Modules in Protein Interaction Networks

PubMed Central

Shen, Hong-Bin

2011-01-01

Modern science of networks has brought significant advances to our understanding of complex systems biology. As a representative model of systems biology, Protein Interaction Networks (PINs) are characterized by a remarkable modular structures, reflecting functional associations between their components. Many methods were proposed to capture cohesive modules so that there is a higher density of edges within modules than those across them. Recent studies reveal that cohesively interacting modules of proteins is not a universal organizing principle in PINs, which has opened up new avenues for revisiting functional modules in PINs. In this paper, functional clusters in PINs are found to be able to form unorthodox structures defined as bi-sparse module. In contrast to the traditional cohesive module, the nodes in the bi-sparse module are sparsely connected internally and densely connected with other bi-sparse or cohesive modules. We present a novel protocol called the BinTree Seeking (BTS) for mining both bi-sparse and cohesive modules in PINs based on Edge Density of Module (EDM) and matrix theory. BTS detects modules by depicting links and nodes rather than nodes alone and its derivation procedure is totally performed on adjacency matrix of networks. The number of modules in a PIN can be automatically determined in the proposed BTS approach. BTS is tested on three real PINs and the results demonstrate that functional modules in PINs are not dominantly cohesive but can be sparse. BTS software and the supporting information are available at: www.csbio.sjtu.edu.cn/bioinf/BTS/. PMID:22140454

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

PubMed

Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

2017-06-21

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7

PubMed Central

Hussain, Rawaa H.; Zawawi, Mariam; Bayfield, Mark A.

2013-01-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3′OH-dependent trailer binding/protection and a UUU-3′OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3′OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily. PMID:23887937

Incorporating modeling and simulations in undergraduate biophysical chemistry course to promote understanding of structure-dynamics-function relationships in proteins.

PubMed

Hati, Sanchita; Bhattacharyya, Sudeep

2016-01-01

A project-based biophysical chemistry laboratory course, which is offered to the biochemistry and molecular biology majors in their senior year, is described. In this course, the classroom study of the structure-function of biomolecules is integrated with the discovery-guided laboratory study of these molecules using computer modeling and simulations. In particular, modern computational tools are employed to elucidate the relationship between structure, dynamics, and function in proteins. Computer-based laboratory protocols that we introduced in three modules allow students to visualize the secondary, super-secondary, and tertiary structures of proteins, analyze non-covalent interactions in protein-ligand complexes, develop three-dimensional structural models (homology model) for new protein sequences and evaluate their structural qualities, and study proteins' intrinsic dynamics to understand their functions. In the fourth module, students are assigned to an authentic research problem, where they apply their laboratory skills (acquired in modules 1-3) to answer conceptual biophysical questions. Through this process, students gain in-depth understanding of protein dynamics-the missing link between structure and function. Additionally, the requirement of term papers sharpens students' writing and communication skills. Finally, these projects result in new findings that are communicated in peer-reviewed journals. © 2016 The International Union of Biochemistry and Molecular Biology.

Decoding telomere protein Rap1: Its telomeric and nontelomeric functions and potential implications in diabetic cardiomyopathy.

PubMed

Cai, Yin; Kandula, Vidya; Kosuru, Ramoji; Ye, Xiaodong; Irwin, Michael G; Xia, Zhengyuan

2017-10-02

Mammalian Rap1, the most conserved telomere-interacting protein, beyond its role within nucleus for the maintenance of telomeric functions, is also well known for its pleiotropic functions in various physiological and pathological conditions associated with metabolism, inflammation and oxidative stress. For all these, nowadays Rap1 is the subject of critical investigations aimed to unveil its molecular signaling pathways and to scrutinize the applicability of its modulation as a promising therapeutic strategy with clinical relevance. However, the underlying intimate mechanisms of Rap1 are not extensively studied, but any modulation of this protein level has been associated with pathologies like inflammation, oxidative stress and deregulated metabolism. This is considerably important in light of the recent discovery of Rap1 modulation in diseases like cancer and cardiac metabolic disorders. In this review, we focus on both the telomeric and nontelomeric functions of Rap1 and its modulation in various health risks, especially on the heart.

Membrane proteins bind lipids selectively to modulate their structure and function.

PubMed

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression

PubMed Central

Stirling, Peter C.; Srayko, Martin; Takhar, Karam S.; Pozniakovsky, Andrei; Hyman, Anthony A.

2007-01-01

The C haperonin Containing Tcp1 (CCT) maintains cellular protein folding homeostasis in the eukaryotic cytosol by assisting the biogenesis of many proteins, including actins, tubulins, and regulators of the cell cycle. Here, we demonstrate that the essential and conserved eukaryotic phosducin-like protein 2 (PhLP2/PLP2) physically interacts with CCT and modulates its folding activity. Consistent with this functional interaction, temperature-sensitive alleles of Saccharomyces cerevisiae PLP2 exhibit cytoskeletal and cell cycle defects. We uncovered several high-copy suppressors of the plp2 alleles, all of which are associated with G1/S cell cycle progression but which do not appreciably affect cytoskeletal protein function or fully rescue the growth defects. Our data support a model in which Plp2p modulates the biogenesis of several CCT substrates relating to cell cycle and cytoskeletal function, which together contribute to the essential function of PLP2. PMID:17429077

Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.

PubMed

Dyson, M H; Rose, S; Mahadevan, L C

2001-08-01

Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone

PubMed Central

Li, Chunmei; Kennedy, Julie; Garcia-Gonzalo, Francesc R.; Romani, Marta; De Mori, Roberta; Bruel, Ange-Line; Gaillard, Dominique; Doray, Bérénice; Lopez, Estelle; Rivière, Jean-Baptiste; Faivre, Laurence; Thauvin-Robinet, Christel; Reiter, Jeremy F.; Blacque, Oliver E.; Valente, Enza Maria; Leroux, Michel R.

2016-01-01

Cilia have a unique diffusion barrier (“gate”) within their proximal region, termed transition zone (TZ), that compartmentalises signalling proteins within the organelle. The TZ is known to harbour two functional modules/complexes (Meckel syndrome [MKS] and Nephronophthisis [NPHP]) defined by genetic interaction, interdependent protein localisation (hierarchy), and proteomic studies. However, the composition and molecular organisation of these modules and their links to human ciliary disease are not completely understood. Here, we reveal Caenorhabditis elegans CEP-290 (mammalian Cep290/Mks4/Nphp6 orthologue) as a central assembly factor that is specific for established MKS module components and depends on the coiled coil region of MKS-5 (Rpgrip1L/Rpgrip1) for TZ localisation. Consistent with a critical role in ciliary gate function, CEP-290 prevents inappropriate entry of membrane-associated proteins into cilia and keeps ARL-13 (Arl13b) from leaking out of cilia via the TZ. We identify a novel MKS module component, TMEM-218 (Tmem218), that requires CEP-290 and other MKS module components for TZ localisation and functions together with the NPHP module to facilitate ciliogenesis. We show that TZ localisation of TMEM-138 (Tmem138) and CDKL-1 (Cdkl1/Cdkl2/Cdkl3/Cdlk4 related), not previously linked to a specific TZ module, similarly depends on CEP-290; surprisingly, neither TMEM-138 or CDKL-1 exhibit interdependent localisation or genetic interactions with core MKS or NPHP module components, suggesting they are part of a distinct, CEP-290-associated module. Lastly, we show that families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) have likely pathogenic mutations in CEP-290-dependent TZ proteins, namely Tmem17, Tmem138, and Tmem231. Notably, patient fibroblasts harbouring mutated Tmem17, a protein not yet ciliopathy-associated, display ciliogenesis defects. Together, our findings expand the repertoire of MKS module-associated proteins—including the previously uncharacterised mammalian Tmem80—and suggest an MKS-5 and CEP-290-dependent assembly pathway for building a functional TZ. PMID:26982032

Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

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

Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia

2014-08-28

The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigatedmore » preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. We identified R. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in R. sphaeroides whereas modules related to RcGTA production and signalling showed lack of preservation in R. sphaeroides. In addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mRNA expression and protein abundance data for which simple correlation measurements have previously had mixed results.« less

Effective comparative analysis of protein-protein interaction networks by measuring the steady-state network flow using a Markov model.

PubMed

Jeong, Hyundoo; Qian, Xiaoning; Yoon, Byung-Jun

2016-10-06

Comparative analysis of protein-protein interaction (PPI) networks provides an effective means of detecting conserved functional network modules across different species. Such modules typically consist of orthologous proteins with conserved interactions, which can be exploited to computationally predict the modules through network comparison. In this work, we propose a novel probabilistic framework for comparing PPI networks and effectively predicting the correspondence between proteins, represented as network nodes, that belong to conserved functional modules across the given PPI networks. The basic idea is to estimate the steady-state network flow between nodes that belong to different PPI networks based on a Markov random walk model. The random walker is designed to make random moves to adjacent nodes within a PPI network as well as cross-network moves between potential orthologous nodes with high sequence similarity. Based on this Markov random walk model, we estimate the steady-state network flow - or the long-term relative frequency of the transitions that the random walker makes - between nodes in different PPI networks, which can be used as a probabilistic score measuring their potential correspondence. Subsequently, the estimated scores can be used for detecting orthologous proteins in conserved functional modules through network alignment. Through evaluations based on multiple real PPI networks, we demonstrate that the proposed scheme leads to improved alignment results that are biologically more meaningful at reduced computational cost, outperforming the current state-of-the-art algorithms. The source code and datasets can be downloaded from http://www.ece.tamu.edu/~bjyoon/CUFID .

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

PubMed Central

2013-01-01

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

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

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

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

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

PubMed Central

2012-01-01

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

CommWalker: correctly evaluating modules in molecular networks in light of annotation bias.

PubMed

Luecken, M D; Page, M J T; Crosby, A J; Mason, S; Reinert, G; Deane, C M

2018-03-15

Detecting novel functional modules in molecular networks is an important step in biological research. In the absence of gold standard functional modules, functional annotations are often used to verify whether detected modules/communities have biological meaning. However, as we show, the uneven distribution of functional annotations means that such evaluation methods favor communities of well-studied proteins. We propose a novel framework for the evaluation of communities as functional modules. Our proposed framework, CommWalker, takes communities as inputs and evaluates them in their local network environment by performing short random walks. We test CommWalker's ability to overcome annotation bias using input communities from four community detection methods on two protein interaction networks. We find that modules accepted by CommWalker are similarly co-expressed as those accepted by current methods. Crucially, CommWalker performs well not only in well-annotated regions, but also in regions otherwise obscured by poor annotation. CommWalker community prioritization both faithfully captures well-validated communities and identifies functional modules that may correspond to more novel biology. The CommWalker algorithm is freely available at opig.stats.ox.ac.uk/resources or as a docker image on the Docker Hub at hub.docker.com/r/lueckenmd/commwalker/. deane@stats.ox.ac.uk. Supplementary data are available at Bioinformatics online.

Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

PubMed Central

Gsponer, Jörg; Babu, M. Madan

2012-01-01

Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

PubMed

Potapov, V; Reichmann, D; Abramovich, R; Filchtinski, D; Zohar, N; Ben Halevy, D; Edelman, M; Sobolev, V; Schreiber, G

2008-12-05

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Function, dynamics and evolution of network motif modules in integrated gene regulatory networks of worm and plant.

PubMed

Defoort, Jonas; Van de Peer, Yves; Vermeirssen, Vanessa

2018-06-05

Gene regulatory networks (GRNs) consist of different molecular interactions that closely work together to establish proper gene expression in time and space. Especially in higher eukaryotes, many questions remain on how these interactions collectively coordinate gene regulation. We study high quality GRNs consisting of undirected protein-protein, genetic and homologous interactions, and directed protein-DNA, regulatory and miRNA-mRNA interactions in the worm Caenorhabditis elegans and the plant Arabidopsis thaliana. Our data-integration framework integrates interactions in composite network motifs, clusters these in biologically relevant, higher-order topological network motif modules, overlays these with gene expression profiles and discovers novel connections between modules and regulators. Similar modules exist in the integrated GRNs of worm and plant. We show how experimental or computational methodologies underlying a certain data type impact network topology. Through phylogenetic decomposition, we found that proteins of worm and plant tend to functionally interact with proteins of a similar age, while at the regulatory level TFs favor same age, but also older target genes. Despite some influence of the duplication mode difference, we also observe at the motif and module level for both species a preference for age homogeneity for undirected and age heterogeneity for directed interactions. This leads to a model where novel genes are added together to the GRNs in a specific biological functional context, regulated by one or more TFs that also target older genes in the GRNs. Overall, we detected topological, functional and evolutionary properties of GRNs that are potentially universal in all species.

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

PubMed

Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

2017-10-01

Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

Photoreactive synthetic regulator of protein function and methods of use thereof

DOEpatents

Trauner, Dirk; Isacoff, Ehud Y; Kramer, Richard H; Banghart, Matthew R; Fortin, Doris L; Mourot, Alexandre

2015-03-31

The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

CytoCluster: A Cytoscape Plugin for Cluster Analysis and Visualization of Biological Networks.

PubMed

Li, Min; Li, Dongyan; Tang, Yu; Wu, Fangxiang; Wang, Jianxin

2017-08-31

Nowadays, cluster analysis of biological networks has become one of the most important approaches to identifying functional modules as well as predicting protein complexes and network biomarkers. Furthermore, the visualization of clustering results is crucial to display the structure of biological networks. Here we present CytoCluster, a cytoscape plugin integrating six clustering algorithms, HC-PIN (Hierarchical Clustering algorithm in Protein Interaction Networks), OH-PIN (identifying Overlapping and Hierarchical modules in Protein Interaction Networks), IPCA (Identifying Protein Complex Algorithm), ClusterONE (Clustering with Overlapping Neighborhood Expansion), DCU (Detecting Complexes based on Uncertain graph model), IPC-MCE (Identifying Protein Complexes based on Maximal Complex Extension), and BinGO (the Biological networks Gene Ontology) function. Users can select different clustering algorithms according to their requirements. The main function of these six clustering algorithms is to detect protein complexes or functional modules. In addition, BinGO is used to determine which Gene Ontology (GO) categories are statistically overrepresented in a set of genes or a subgraph of a biological network. CytoCluster can be easily expanded, so that more clustering algorithms and functions can be added to this plugin. Since it was created in July 2013, CytoCluster has been downloaded more than 9700 times in the Cytoscape App store and has already been applied to the analysis of different biological networks. CytoCluster is available from http://apps.cytoscape.org/apps/cytocluster.

CytoCluster: A Cytoscape Plugin for Cluster Analysis and Visualization of Biological Networks

PubMed Central

Li, Min; Li, Dongyan; Tang, Yu; Wang, Jianxin

2017-01-01

Nowadays, cluster analysis of biological networks has become one of the most important approaches to identifying functional modules as well as predicting protein complexes and network biomarkers. Furthermore, the visualization of clustering results is crucial to display the structure of biological networks. Here we present CytoCluster, a cytoscape plugin integrating six clustering algorithms, HC-PIN (Hierarchical Clustering algorithm in Protein Interaction Networks), OH-PIN (identifying Overlapping and Hierarchical modules in Protein Interaction Networks), IPCA (Identifying Protein Complex Algorithm), ClusterONE (Clustering with Overlapping Neighborhood Expansion), DCU (Detecting Complexes based on Uncertain graph model), IPC-MCE (Identifying Protein Complexes based on Maximal Complex Extension), and BinGO (the Biological networks Gene Ontology) function. Users can select different clustering algorithms according to their requirements. The main function of these six clustering algorithms is to detect protein complexes or functional modules. In addition, BinGO is used to determine which Gene Ontology (GO) categories are statistically overrepresented in a set of genes or a subgraph of a biological network. CytoCluster can be easily expanded, so that more clustering algorithms and functions can be added to this plugin. Since it was created in July 2013, CytoCluster has been downloaded more than 9700 times in the Cytoscape App store and has already been applied to the analysis of different biological networks. CytoCluster is available from http://apps.cytoscape.org/apps/cytocluster. PMID:28858211

Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein.

PubMed

Banerjee, Paromita; Schoenfeld, Brian P; Bell, Aaron J; Choi, Catherine H; Bradley, Michael P; Hinchey, Paul; Kollaros, Maria; Park, Jae H; McBride, Sean M J; Dockendorff, Thomas C

2010-05-12

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

Asprosin, a fasting-induced glucogenic protein hormone

USDA-ARS?s Scientific Manuscript database

Hepatic glucose release into the circulation is vital for brain function and survival during periods of fasting and is modulated by an array of hormones that precisely regulate plasma glucose levels. We have identified a fasting-induced protein hormone that modulates hepatic glucose release. It is t...

MINE: Module Identification in Networks

PubMed Central

2011-01-01

Background Graphical models of network associations are useful for both visualizing and integrating multiple types of association data. Identifying modules, or groups of functionally related gene products, is an important challenge in analyzing biological networks. However, existing tools to identify modules are insufficient when applied to dense networks of experimentally derived interaction data. To address this problem, we have developed an agglomerative clustering method that is able to identify highly modular sets of gene products within highly interconnected molecular interaction networks. Results MINE outperforms MCODE, CFinder, NEMO, SPICi, and MCL in identifying non-exclusive, high modularity clusters when applied to the C. elegans protein-protein interaction network. The algorithm generally achieves superior geometric accuracy and modularity for annotated functional categories. In comparison with the most closely related algorithm, MCODE, the top clusters identified by MINE are consistently of higher density and MINE is less likely to designate overlapping modules as a single unit. MINE offers a high level of granularity with a small number of adjustable parameters, enabling users to fine-tune cluster results for input networks with differing topological properties. Conclusions MINE was created in response to the challenge of discovering high quality modules of gene products within highly interconnected biological networks. The algorithm allows a high degree of flexibility and user-customisation of results with few adjustable parameters. MINE outperforms several popular clustering algorithms in identifying modules with high modularity and obtains good overall recall and precision of functional annotations in protein-protein interaction networks from both S. cerevisiae and C. elegans. PMID:21605434

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Schäfer, Bettina; Philipp, Ute; Kuiper, Heidi; Leeb, Tosso; Mehta, Meenal; Kirchhoff, Christiane; Töpfer-Petersen, Edda

2007-05-01

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

NASA Astrophysics Data System (ADS)

Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

2016-04-01

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

Botulinum neurotoxin: a marvel of protein design.

PubMed

Montal, Mauricio

2010-01-01

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

Controlling allosteric networks in proteins

NASA Astrophysics Data System (ADS)

Dokholyan, Nikolay

2013-03-01

We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

PubMed Central

Walther, Cornelia

2015-01-01

The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

GIV/Girdin activates Gαi and inhibits Gαs via the same motif

PubMed Central

Gupta, Vijay; Bhandari, Deepali; Leyme, Anthony; Aznar, Nicolas; Midde, Krishna K.; Lo, I-Chung; Ear, Jason; Niesman, Ingrid; López-Sánchez, Inmaculada; Blanco-Canosa, Juan Bautista; von Zastrow, Mark; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

2016-01-01

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses. PMID:27621449

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

PubMed Central

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

2015-01-01

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

Physiological functions of MTA family of proteins.

PubMed

Sen, Nirmalya; Gui, Bin; Kumar, Rakesh

2014-12-01

Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of CaV Function

PubMed Central

2017-01-01

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design. PMID:28278376

Molecular mechanics and dynamics characterization of an in silico mutated protein: a stand-alone lab module or support activity for in vivo and in vitro analyses of targeted proteins.

PubMed

Chiang, Harry; Robinson, Lucy C; Brame, Cynthia J; Messina, Troy C

2013-01-01

Over the past 20 years, the biological sciences have increasingly incorporated chemistry, physics, computer science, and mathematics to aid in the development and use of mathematical models. Such combined approaches have been used to address problems from protein structure-function relationships to the workings of complex biological systems. Computer simulations of molecular events can now be accomplished quickly and with standard computer technology. Also, simulation software is freely available for most computing platforms, and online support for the novice user is ample. We have therefore created a molecular dynamics laboratory module to enhance undergraduate student understanding of molecular events underlying organismal phenotype. This module builds on a previously described project in which students use site-directed mutagenesis to investigate functions of conserved sequence features in members of a eukaryotic protein kinase family. In this report, we detail the laboratory activities of a MD module that provide a complement to phenotypic outcomes by providing a hypothesis-driven and quantifiable measure of predicted structural changes caused by targeted mutations. We also present examples of analyses students may perform. These laboratory activities can be integrated with genetics or biochemistry experiments as described, but could also be used independently in any course that would benefit from a quantitative approach to protein structure-function relationships. Copyright © 2013 Wiley Periodicals, Inc.

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

PubMed Central

Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

2013-01-01

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

PubMed

Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.

A bioarchitectonic approach to the modular engineering of metabolism.

PubMed

Kerfeld, Cheryl A

2017-09-26

Dissociating the complexity of metabolic processes into modules is a shift in focus from the single gene/gene product to functional and evolutionary units spanning the scale of biological organization. When viewing the levels of biological organization through this conceptual lens, modules are found across the continuum: domains within proteins, co-regulated groups of functionally associated genes, operons, metabolic pathways and (sub)cellular compartments. Combining modules as components or subsystems of a larger system typically leads to increased complexity and the emergence of new functions. By virtue of their potential for 'plug and play' into new contexts, modules can be viewed as units of both evolution and engineering. Through consideration of lessons learned from recent efforts to install new metabolic modules into cells and the emerging understanding of the structure, function and assembly of protein-based organelles, bacterial microcompartments, a structural bioengineering approach is described: one that builds from an architectural vocabulary of protein domains. This bioarchitectonic approach to engineering cellular metabolism can be applied to microbial cell factories, used in the programming of members of synthetic microbial communities or used to attain additional levels of metabolic organization in eukaryotic cells for increasing primary productivity and as the foundation of a green economy.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Author(s).

ASD: a comprehensive database of allosteric proteins and modulators

PubMed Central

Huang, Zhimin; Zhu, Liang; Cao, Yan; Wu, Geng; Liu, Xinyi; Chen, Yingyi; Wang, Qi; Shi, Ting; Zhao, Yaxue; Wang, Yuefei; Li, Weihua; Li, Yixue; Chen, Haifeng; Chen, Guoqiang; Zhang, Jian

2011-01-01

Allostery is the most direct, rapid and efficient way of regulating protein function, ranging from the control of metabolic mechanisms to signal-transduction pathways. However, an enormous amount of unsystematic allostery information has deterred scientists who could benefit from this field. Here, we present the AlloSteric Database (ASD), the first online database that provides a central resource for the display, search and analysis of structure, function and related annotation for allosteric molecules. Currently, ASD contains 336 allosteric proteins from 101 species and 8095 modulators in three categories (activators, inhibitors and regulators). Proteins are annotated with a detailed description of allostery, biological process and related diseases, and modulators with binding affinity, physicochemical properties and therapeutic area. Integrating the information of allosteric proteins in ASD should allow for the identification of specific allosteric sites of a given subtype among proteins of the same family that can potentially serve as ideal targets for experimental validation. In addition, modulators curated in ASD can be used to investigate potent allosteric targets for the query compound, and also help chemists to implement structure modifications for novel allosteric drug design. Therefore, ASD could be a platform and a starting point for biologists and medicinal chemists for furthering allosteric research. ASD is freely available at http://mdl.shsmu.edu.cn/ASD/. PMID:21051350

An efficient algorithm for pairwise local alignment of protein interaction networks

DOE PAGES

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

2015-04-01

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

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.

PubMed

Senetar, Melissa A; Foster, Stanley J; McCann, Richard O

2004-12-14

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

PubMed

Branham-O'Connor, Melissa; Robichaux, William G; Zhang, Xian-Kui; Cho, Hyeseon; Kehrl, John H; Lanier, Stephen M; Blumer, Joe B

2014-04-11

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

TALE transcription factors during early development of the vertebrate brain and eye.

PubMed

Schulte, Dorothea; Frank, Dale

2014-01-01

Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is, therefore, given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification. Copyright © 2013 Wiley Periodicals, Inc.

DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

PubMed Central

Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

1999-01-01

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

Interactions Between Flavonoid-Rich Extracts and Sodium Caseinate Modulate Protein Functionality and Flavonoid Bioaccessibility in Model Food Systems.

PubMed

Elegbede, Jennifer L; Li, Min; Jones, Owen G; Campanella, Osvaldo H; Ferruzzi, Mario G

2018-05-01

With growing interest in formulating new food products with added protein and flavonoid-rich ingredients for health benefits, direct interactions between these ingredient classes becomes critical in so much as they may impact protein functionality, product quality, and flavonoids bioavailability. In this study, sodium caseinate (SCN)-based model products (foams and emulsions) were formulated with grape seed extract (GSE, rich in galloylated flavonoids) and green tea extract (GTE, rich in nongalloylated flavonoids), respectively, to assess changes in functional properties of SCN and impacts on flavonoid bioaccessibility. Experiments with pure flavonoids suggested that galloylated flavonoids reduced air-water interfacial tension of 0.01% SCN dispersions more significantly than nongalloylated flavonoids at high concentrations (>50 μg/mL). This observation was supported by changes in stability of 5% SCN foam, which showed that foam stability was increased at high levels of GSE (≥50 μg/mL, P < 0.05) but was not affected by GTE. However, flavonoid extracts had modest effects on SCN emulsion. In addition, galloylated flavonoids had higher bioaccessibility in both SCN foam and emulsion. These results suggest that SCN-flavonoid binding interactions can modulate protein functionality leading to difference in performance and flavonoid bioaccessibility of protein-based products. As information on the beneficial health effects of flavonoids expands, it is likely that usage of these ingredients in consumer foods will increase. However, the necessary levels to provide such benefits may exceed those that begin to impact functionality of the macronutrients such as proteins. Flavonoid inclusion within protein matrices may modulate protein functionality in a food system and modify critical consumer traits or delivery of these beneficial plant-derived components. The product matrices utilized in this study offer relevant model systems to evaluate how fortification with flavonoid-rich extracts allows for differing effects on formability and stability of the protein-based systems, and on bioaccessibility of fortified flavonoid extracts. © 2018 Institute of Food Technologists®.

Molecular tweezers modulate 14-3-3 protein-protein interactions

NASA Astrophysics Data System (ADS)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Garlic compounds modulate macrophage and T-lymphocyte functions.

PubMed

Lau, B H; Yamasaki, T; Gridley, D S

1991-06-01

Organosulfur compounds of garlic have been shown to inhibit growth of animal tumors and to modulate the activity of diverse chemical carcinogens. There is also evidence that garlic may modulate antitumor immunity. In this study, we determined the effects of an aqueous garlic extract and a protein fraction isolated from the extract on the chemiluminescent oxidative burst of the murine J774 macrophage cell line and thioglycollate-elicited peritoneal macrophages obtained from BALB/c mice. T-lymphocyte activity was determined using mouse splenocytes incubated with phytohemagglutinin, labeled with [3H]-thymidine and assayed for lymphoproliferation. Significant dose-related augmentation of oxidative burst was observed with garlic extract and the protein fraction. The protein fraction also enhanced the T-lymphocyte blastogenesis. The data suggest that garlic compounds may serve as biological response modifiers by augmenting macrophage and T-lymphocyte functions.

JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

PubMed

Dutta, Aditya; Choudhary, Pratibha; Caruana, Julie; Raina, Ramesh

2017-09-01

Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

PubMed

Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

2002-09-01

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

Huntingtin-interacting protein 1 influences worm and mouse presynaptic function and protects Caenorhabditis elegans neurons against mutant polyglutamine toxicity.

PubMed

Parker, J Alex; Metzler, Martina; Georgiou, John; Mage, Marilyne; Roder, John C; Rose, Ann M; Hayden, Michael R; Néri, Christian

2007-10-10

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntington's disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD. We report that the Caenorhabditis elegans HIP1 homolog hipr-1 modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1-/- mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced by hipr-1 loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such as unc-11/AP180, unc-26/synaptojanin, and unc-57/endophilin. Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.

Efficient and accurate Greedy Search Methods for mining functional modules in protein interaction networks.

PubMed

He, Jieyue; Li, Chaojun; Ye, Baoliu; Zhong, Wei

2012-06-25

Most computational algorithms mainly focus on detecting highly connected subgraphs in PPI networks as protein complexes but ignore their inherent organization. Furthermore, many of these algorithms are computationally expensive. However, recent analysis indicates that experimentally detected protein complexes generally contain Core/attachment structures. In this paper, a Greedy Search Method based on Core-Attachment structure (GSM-CA) is proposed. The GSM-CA method detects densely connected regions in large protein-protein interaction networks based on the edge weight and two criteria for determining core nodes and attachment nodes. The GSM-CA method improves the prediction accuracy compared to other similar module detection approaches, however it is computationally expensive. Many module detection approaches are based on the traditional hierarchical methods, which is also computationally inefficient because the hierarchical tree structure produced by these approaches cannot provide adequate information to identify whether a network belongs to a module structure or not. In order to speed up the computational process, the Greedy Search Method based on Fast Clustering (GSM-FC) is proposed in this work. The edge weight based GSM-FC method uses a greedy procedure to traverse all edges just once to separate the network into the suitable set of modules. The proposed methods are applied to the protein interaction network of S. cerevisiae. Experimental results indicate that many significant functional modules are detected, most of which match the known complexes. Results also demonstrate that the GSM-FC algorithm is faster and more accurate as compared to other competing algorithms. Based on the new edge weight definition, the proposed algorithm takes advantages of the greedy search procedure to separate the network into the suitable set of modules. Experimental analysis shows that the identified modules are statistically significant. The algorithm can reduce the computational time significantly while keeping high prediction accuracy.

Modulators of 14-3-3 Protein–Protein Interactions

PubMed Central

2017-01-01

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein–protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as “undruggable” targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products. PMID:28968506

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans

PubMed Central

Chan, Shih-Peng; Slack, Frank J.

2009-01-01

The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3′UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs. PMID:19627982

A global interaction network maps a wiring diagram of cellular function

PubMed Central

Costanzo, Michael; VanderSluis, Benjamin; Koch, Elizabeth N.; Baryshnikova, Anastasia; Pons, Carles; Tan, Guihong; Wang, Wen; Usaj, Matej; Hanchard, Julia; Lee, Susan D.; Pelechano, Vicent; Styles, Erin B.; Billmann, Maximilian; van Leeuwen, Jolanda; van Dyk, Nydia; Lin, Zhen-Yuan; Kuzmin, Elena; Nelson, Justin; Piotrowski, Jeff S.; Srikumar, Tharan; Bahr, Sondra; Chen, Yiqun; Deshpande, Raamesh; Kurat, Christoph F.; Li, Sheena C.; Li, Zhijian; Usaj, Mojca Mattiazzi; Okada, Hiroki; Pascoe, Natasha; Luis, Bryan-Joseph San; Sharifpoor, Sara; Shuteriqi, Emira; Simpkins, Scott W.; Snider, Jamie; Suresh, Harsha Garadi; Tan, Yizhao; Zhu, Hongwei; Malod-Dognin, Noel; Janjic, Vuk; Przulj, Natasa; Troyanskaya, Olga G.; Stagljar, Igor; Xia, Tian; Ohya, Yoshikazu; Gingras, Anne-Claude; Raught, Brian; Boutros, Michael; Steinmetz, Lars M.; Moore, Claire L.; Rosebrock, Adam P.; Caudy, Amy A.; Myers, Chad L.; Andrews, Brenda; Boone, Charles

2017-01-01

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing over 23 million double mutants, identifying ~550,000 negative and ~350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell. PMID:27708008

CELFish ways to modulate mRNA decay

PubMed Central

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

PubMed

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Inborn errors of metabolism and the human interactome: a systems medicine approach.

PubMed

Woidy, Mathias; Muntau, Ania C; Gersting, Søren W

2018-02-05

The group of inborn errors of metabolism (IEM) displays a marked heterogeneity and IEM can affect virtually all functions and organs of the human organism; however, IEM share that their associated proteins function in metabolism. Most proteins carry out cellular functions by interacting with other proteins, and thus are organized in biological networks. Therefore, diseases are rarely the consequence of single gene mutations but of the perturbations caused in the related cellular network. Systematic approaches that integrate multi-omics and database information into biological networks have successfully expanded our knowledge of complex disorders but network-based strategies have been rarely applied to study IEM. We analyzed IEM on a proteome scale and found that IEM-associated proteins are organized as a network of linked modules within the human interactome of protein interactions, the IEM interactome. Certain IEM disease groups formed self-contained disease modules, which were highly interlinked. On the other hand, we observed disease modules consisting of proteins from many different disease groups in the IEM interactome. Moreover, we explored the overlap between IEM and non-IEM disease genes and applied network medicine approaches to investigate shared biological pathways, clinical signs and symptoms, and links to drug targets. The provided resources may help to elucidate the molecular mechanisms underlying new IEM, to uncover the significance of disease-associated mutations, to identify new biomarkers, and to develop novel therapeutic strategies.

Exosomes released by keratinocytes modulate melanocyte pigmentation

PubMed Central

Cicero, Alessandra Lo; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

2015-01-01

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

Ras-sensitive IMP modulation of the Raf/MEK/ERK cascade through KSR1.

PubMed

Matheny, Sharon A; White, Michael A

2006-01-01

The E3 ubiquitin ligase IMP (impedes mitogenic signal propagation) was isolated as a novel Ras effector that negatively regulates ERK1/2 activation. Current evidence suggests that IMP limits the functional assembly of Raf/MEK complexes by inactivation of the KSR1 adaptor/scaffold protein. Interaction with Ras-GTP stimulates IMP autoubiquitination to relieve limitations on KSR function. The elevated sensitivity of IMP-depleted cells to ERK1/2 pathway activation suggests IMP acts as a signal threshold regulator by imposing reversible restrictions on the assembly of functional Raf/MEK/ERK kinase modules. These observations challenge commonly held concepts of signal transmission by Ras to the MAPK pathway and provide evidence for the role of amplitude modulation in tuning cellular responses to ERK1/2 pathway engagement. Here we describe details of the methods, including RNA interference, ubiquitin ligase assays, and protein complex analysis, that can be used to display the Ras-sensitive contribution of IMP to KSR-dependent modulation of the Raf/MEK/ERK pathway.

Biphasic patterns of diversification and the emergence of modules

PubMed Central

Mittenthal, Jay; Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

2012-01-01

The intricate molecular and cellular structure of organisms converts energy to work, which builds and maintains structure. Evolving structure implements modules, in which parts are tightly linked. Each module performs characteristic functions. In this work we propose that a module can emerge through two phases of diversification of parts. Early in the first phase of this biphasic pattern, the parts have weak linkage—they interact weakly and associate variously. The parts diversify and compete. Under selection for performance, interactions among the parts increasingly constrain their structure and associations. As many variants are eliminated, parts self-organize into modules with tight linkage. Linkage may increase in response to exogenous stresses as well as endogenous processes. In the second phase of diversification, variants of the module and its functions evolve and become new parts for a new cycle of generation of higher-level modules. This linkage hypothesis can interpret biphasic patterns in the diversification of protein domain structure, RNA and protein shapes, and networks in metabolism, codes, and embryos, and can explain hierarchical levels of structural organization that are widespread in biology. PMID:22891076

Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

PubMed Central

Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

2011-01-01

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection. PMID:21559274

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

PubMed Central

Lovero, Kathryn L.; Fukata, Yuko; Granger, Adam J.; Fukata, Masaki; Nicoll, Roger A.

2015-01-01

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95. PMID:26178195

An ensemble framework for clustering protein-protein interaction networks.

PubMed

Asur, Sitaram; Ucar, Duygu; Parthasarathy, Srinivasan

2007-07-01

Protein-Protein Interaction (PPI) networks are believed to be important sources of information related to biological processes and complex metabolic functions of the cell. The presence of biologically relevant functional modules in these networks has been theorized by many researchers. However, the application of traditional clustering algorithms for extracting these modules has not been successful, largely due to the presence of noisy false positive interactions as well as specific topological challenges in the network. In this article, we propose an ensemble clustering framework to address this problem. For base clustering, we introduce two topology-based distance metrics to counteract the effects of noise. We develop a PCA-based consensus clustering technique, designed to reduce the dimensionality of the consensus problem and yield informative clusters. We also develop a soft consensus clustering variant to assign multifaceted proteins to multiple functional groups. We conduct an empirical evaluation of different consensus techniques using topology-based, information theoretic and domain-specific validation metrics and show that our approaches can provide significant benefits over other state-of-the-art approaches. Our analysis of the consensus clusters obtained demonstrates that ensemble clustering can (a) produce improved biologically significant functional groupings; and (b) facilitate soft clustering by discovering multiple functional associations for proteins. Supplementary data are available at Bioinformatics online.

Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus

PubMed Central

Zhang, Jiyong; Sadowska, Grazyna B.; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A.; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Stonestreet, Barbara S.

2015-01-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti–IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti–IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti–IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.-E., Bodge, C. A., Cummings, E., Lim, Y.-P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus. PMID:25609424

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology.

PubMed

Kiang, J G; Tsokos, G C

1998-11-01

Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic. In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults. Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer. Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases. Most of the HSPs interact with other proteins in cells and alter their function. These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions. In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways. The clinical importance and therapeutic potential of HSPs are discussed.

A Proteome-wide Domain-centric Perspective on Protein Phosphorylation *

PubMed Central

Palmeri, Antonio; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela; Gherardini, Pier Federico

2014-01-01

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area. PMID:24830415

Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

PubMed

Kessels, Michael M; Qualmann, Britta

2015-09-01

A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

Applied Graph-Mining Algorithms to Study Biomolecular Interaction Networks

PubMed Central

2014-01-01

Protein-protein interaction (PPI) networks carry vital information on the organization of molecular interactions in cellular systems. The identification of functionally relevant modules in PPI networks is one of the most important applications of biological network analysis. Computational analysis is becoming an indispensable tool to understand large-scale biomolecular interaction networks. Several types of computational methods have been developed and employed for the analysis of PPI networks. Of these computational methods, graph comparison and module detection are the two most commonly used strategies. This review summarizes current literature on graph kernel and graph alignment methods for graph comparison strategies, as well as module detection approaches including seed-and-extend, hierarchical clustering, optimization-based, probabilistic, and frequent subgraph methods. Herein, we provide a comprehensive review of the major algorithms employed under each theme, including our recently published frequent subgraph method, for detecting functional modules commonly shared across multiple cancer PPI networks. PMID:24800226

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

PubMed

Harms, Alexander; Brodersen, Ditlev Egeskov; Mitarai, Namiko; Gerdes, Kenn

2018-06-07

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules. Copyright © 2018 Elsevier Inc. All rights reserved.

QQS orphan gene regulates carbon and nitrogen partitioning across species via NF-YC interactions

PubMed Central

Li, Ling; Zheng, Wenguang; Zhu, Yanbing; Ye, Huaxun; Tang, Buyun; Arendsee, Zebulun W.; Jones, Dallas; Li, Ruoran; Ortiz, Diego; Zhao, Xuefeng; Du, Chuanlong; Nettleton, Dan; Scott, M. Paul; Salas-Fernandez, Maria G.; Yin, Yanhai; Wurtele, Eve Syrkin

2015-01-01

The allocation of carbon and nitrogen resources to the synthesis of plant proteins, carbohydrates, and lipids is complex and under the control of many genes; much remains to be understood about this process. QQS (Qua-Quine Starch; At3g30720), an orphan gene unique to Arabidopsis thaliana, regulates metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, modulating leaf and seed composition in Arabidopsis and soybean. Here the universality of QQS function in modulating carbon and nitrogen allocation is exemplified by a series of transgenic experiments. We show that ectopic expression of QQS increases soybean protein independent of the genetic background and original protein content of the cultivar. Furthermore, transgenic QQS expression increases the protein content of maize, a C4 species (a species that uses 4-carbon photosynthesis), and rice, a protein-poor agronomic crop, both highly divergent from Arabidopsis. We determine that QQS protein binds to the transcriptional regulator AtNF-YC4 (Arabidopsis nuclear factor Y, subunit C4). Overexpression of AtNF-YC4 in Arabidopsis mimics the QQS-overexpression phenotype, increasing protein and decreasing starch levels. NF-YC, a component of the NF-Y complex, is conserved across eukaryotes. The NF-YC4 homologs of soybean, rice, and maize also bind to QQS, which provides an explanation of how QQS can act in species where it does not occur endogenously. These findings are, to our knowledge, the first insight into the mechanism of action of QQS in modulating carbon and nitrogen allocation across species. They have major implications for the emergence and function of orphan genes, and identify a nontransgenic strategy for modulating protein levels in crop species, a trait of great agronomic significance. PMID:26554020

QQS orphan gene regulates carbon and nitrogen partitioning across species via NF-YC interactions.

PubMed

Li, Ling; Zheng, Wenguang; Zhu, Yanbing; Ye, Huaxun; Tang, Buyun; Arendsee, Zebulun W; Jones, Dallas; Li, Ruoran; Ortiz, Diego; Zhao, Xuefeng; Du, Chuanlong; Nettleton, Dan; Scott, M Paul; Salas-Fernandez, Maria G; Yin, Yanhai; Wurtele, Eve Syrkin

2015-11-24

The allocation of carbon and nitrogen resources to the synthesis of plant proteins, carbohydrates, and lipids is complex and under the control of many genes; much remains to be understood about this process. QQS (Qua-Quine Starch; At3g30720), an orphan gene unique to Arabidopsis thaliana, regulates metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, modulating leaf and seed composition in Arabidopsis and soybean. Here the universality of QQS function in modulating carbon and nitrogen allocation is exemplified by a series of transgenic experiments. We show that ectopic expression of QQS increases soybean protein independent of the genetic background and original protein content of the cultivar. Furthermore, transgenic QQS expression increases the protein content of maize, a C4 species (a species that uses 4-carbon photosynthesis), and rice, a protein-poor agronomic crop, both highly divergent from Arabidopsis. We determine that QQS protein binds to the transcriptional regulator AtNF-YC4 (Arabidopsis nuclear factor Y, subunit C4). Overexpression of AtNF-YC4 in Arabidopsis mimics the QQS-overexpression phenotype, increasing protein and decreasing starch levels. NF-YC, a component of the NF-Y complex, is conserved across eukaryotes. The NF-YC4 homologs of soybean, rice, and maize also bind to QQS, which provides an explanation of how QQS can act in species where it does not occur endogenously. These findings are, to our knowledge, the first insight into the mechanism of action of QQS in modulating carbon and nitrogen allocation across species. They have major implications for the emergence and function of orphan genes, and identify a nontransgenic strategy for modulating protein levels in crop species, a trait of great agronomic significance.

Regulation, Signaling, and Physiological Functions of G-Proteins.

PubMed

Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

2016-09-25

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator).

PubMed

Godwin, Ryan C; Melvin, Ryan L; Gmeiner, William H; Salsbury, Freddie R

2017-01-31

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein-protein or protein-DNA interactions.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

PubMed

Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

2016-10-01

Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules.

PubMed

An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin

2015-10-01

Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor gamma through protein-protein interaction.

PubMed

Choi, Youn-Hee; Kim, Ha-il; Seong, Je Kyung; Yu, Dae-Yeul; Cho, Hyeseong; Lee, Mi-Ock; Lee, Jae Myun; Ahn, Yong-ho; Kim, Se Jong; Park, Jeon Han

2004-01-16

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

Analysis of sDMA modifications of PIWI proteins

PubMed Central

Honda, Shozo; Kirino, Yoriko; Kirino, Yohei

2015-01-01

Summary Arginine methylation is an important post-translational protein modification that modulates protein function for a wide range of biological processes. PIWI proteins, a subclade of the Argonaute family proteins, contain evolutionarily conserved symmetrical dimethylarginines (sDMAs). It has become increasingly apparent that the sDMAs of PIWI proteins serve as binding elements for TUDOR-domain containing proteins and that sDMA-dependent protein interactions play crucial roles in the biogenesis and function of PIWI-interacting RNAs (piRNAs). We describe a method for detecting PIWI sDMAs and purifying PIWI/piRNA complexes using anti-sDMA antibodies. PMID:24178562

Inferring genome-wide functional modulatory network: a case study on NF-κB/RelA transcription factor.

PubMed

Li, Xueling; Zhu, Min; Brasier, Allan R; Kudlicki, Andrzej S

2015-04-01

How different pathways lead to the activation of a specific transcription factor (TF) with specific effects is not fully understood. We model context-specific transcriptional regulation as a modulatory network: triplets composed of a TF, target gene, and modulator. Modulators usually affect the activity of a specific TF at the posttranscriptional level in a target gene-specific action mode. This action may be classified as enhancement, attenuation, or inversion of either activation or inhibition. As a case study, we inferred, from a large collection of expression profiles, all potential modulations of NF-κB/RelA. The predicted modulators include many proteins previously not reported as physically binding to RelA but with relevant functions, such as RNA processing, cell cycle, mitochondrion, ubiquitin-dependent proteolysis, and chromatin modification. Modulators from different processes exert specific prevalent action modes on distinct pathways. Modulators from noncoding RNA, RNA-binding proteins, TFs, and kinases modulate the NF-κB/RelA activity with specific action modes consistent with their molecular functions and modulation level. The modulatory networks of NF-κB/RelA in the context epithelial-mesenchymal transition (EMT) and burn injury have different modulators, including those involved in extracellular matrix (FBN1), cytoskeletal regulation (ACTN1), and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long intergenic nonprotein coding RNA, and tumor suppression (FOXP1) for EMT, and TXNIP, GAPDH, PKM2, IFIT5, LDHA, NID1, and TPP1 for burn injury.

Advancing clinical development pathways for new CFTR modulators in cystic fibrosis.

PubMed

Mayer-Hamblett, Nicole; Boyle, Michael; VanDevanter, Donald

2016-05-01

Cystic fibrosis (CF) is a life-shortening genetic disease affecting approximately 70,000 individuals worldwide. Until recently, drug development efforts have emphasised therapies treating downstream signs and symptoms resulting from the underlying CF biological defect: reduced function of the CF transmembrane conductance regulator (CFTR) protein. The current CF drug development landscape has expanded to include therapies that enhance CFTR function by either restoring wild-type CFTR protein expression or increasing (modulating) the function of mutant CFTR proteins in cells. To date, two systemic small-molecule CFTR modulators have been evaluated in pivotal clinical trials in individuals with CF and specific mutant CFTR genotypes that have led to regulatory review and/or approval. Advances in the discovery of CFTR modulators as a promising new class of therapies have been impressive, yet work remains to develop highly effective, disease-modifying modulators for individuals of all CF genotypes. The objectives of this review are to outline the challenges and opportunities in drug development created by systemic genotype-specific CFTR modulators, highlight the advantages of sweat chloride as an established biomarker of CFTR activity to streamline early-phase development and summarise options for later phase clinical trial designs that respond to the adoption of approved genotype-specific modulators into standard of care. An optimal development framework will be needed to move the most promising therapies efficiently through the drug development pipeline and ultimately deliver efficacious and safe therapies to all individuals with CF. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Harnessing Drug Resistance: Using ABC Transporter Proteins To Target Cancer Cells

PubMed Central

Leitner, Heather M.; Kachadourian, Remy; Day, Brian J.

2007-01-01

The ATP-binding cassette (ABC) class of proteins is one of the most functionally diverse transporter families found in biological systems. Although the abundance of ABC proteins varies between species, they are highly conserved in sequence and often demonstrate similar functions across prokaryotic and eukaryotic organisms. Beginning with a brief summary of the events leading to our present day knowledge of ABC transporters, the purpose of this review is to discuss the potential for utilizing ABC transporters as a means for cellular glutathione (GSH) modulation. GSH is one of the most abundant thiol antioxidants in cells. It is involved in cellular division, protein and DNA synthesis, maintenance of cellular redox status and xenobiotic metabolism. Cellular GSH levels are often altered in many disease states including cancer. Over the past two decades there has been considerable emphasis on methods to sensitize cancer cells to chemotherapeutics and ionization radiation therapy by GSH depletion. We contend that ABC transporters, particularly multi-drug resistant proteins (MRPs), may be used as therapeutic targets for applications aimed at modulation of GSH levels. This review will emphasize MRP-mediated modulation of intracellular GSH levels as a potential alternative and adjunctive approach for cancer therapy. PMID:17585883

Identification and functional analysis of a core gene module associated with hepatitis C virus-induced human hepatocellular carcinoma progression.

PubMed

Bai, Gaobo; Zheng, Wenling; Ma, Wenli

2018-05-01

Hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC) progression may be due to a complex multi-step processes. The developmental mechanism of these processes is worth investigating for the prevention, diagnosis and therapy of HCC. The aim of the present study was to investigate the molecular mechanism underlying the progression of HCV-induced hepatocarcinogenesis. First, the dynamic gene module, consisting of key genes associated with progression between the normal stage and HCC, was identified using the Weighted Gene Co-expression Network Analysis tool from R language. By defining those genes in the module as seeds, the change of co-expression in differentially expressed gene sets in two consecutive stages of pathological progression was examined. Finally, interaction pairs of HCV viral proteins and their directly targeted proteins in the identified module were extracted from the literature and a comprehensive interaction dataset from yeast two-hybrid experiments. By combining the interactions between HCV and their targets, and protein-protein interactions in the Search Tool for the Retrieval of Interacting Genes database (STRING), the HCV-key genes interaction network was constructed and visualized using Cytoscape software 3.2. As a result, a module containing 44 key genes was identified to be associated with HCC progression, due to the dynamic features and functions of those genes in the module. Several important differentially co-expressed gene pairs were identified between non-HCC and HCC stages. In the key genes, cyclin dependent kinase 1 (CDK1), NDC80, cyclin A2 (CCNA2) and rac GTPase activating protein 1 (RACGAP1) were shown to be targeted by the HCV nonstructural proteins NS5A, NS3 and NS5B, respectively. The four genes perform an intermediary role between the HCV viral proteins and the dysfunctional module in the HCV key genes interaction network. These findings provided valuable information for understanding the mechanism of HCV-induced HCC progression and for seeking drug targets for the therapy and prevention of HCC.

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions.

PubMed

Zehender, Hartmut; Mayr, Lorenz M

2007-10-01

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.

Exploring Protein Structure and Dynamics through a Project-Oriented Biochemistry Laboratory Module

ERIC Educational Resources Information Center

Lipchock, James M.; Ginther, Patrick S.; Douglas, Bonnie B.; Bird, Kelly E.; Loria, J. Patrick

2017-01-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant…

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

PubMed Central

Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

2012-01-01

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

Toward high-resolution computational design of helical membrane protein structure and function

PubMed Central

Barth, Patrick; Senes, Alessandro

2016-01-01

The computational design of α-helical membrane proteins is still in its infancy but has made important progress. De novo design has produced stable, specific and active minimalistic oligomeric systems. Computational re-engineering can improve stability and modulate the function of natural membrane proteins. Currently, the major hurdle for the field is not computational, but the experimental characterization of the designs. The emergence of new structural methods for membrane proteins will accelerate progress PMID:27273630

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.

Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

PubMed Central

Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

2015-01-01

Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

Identification of Unstable Network Modules Reveals Disease Modules Associated with the Progression of Alzheimer’s Disease

PubMed Central

Kikuchi, Masataka; Ogishima, Soichi; Miyamoto, Tadashi; Miyashita, Akinori; Kuwano, Ryozo; Nakaya, Jun; Tanaka, Hiroshi

2013-01-01

Alzheimer’s disease (AD), the most common cause of dementia, is associated with aging, and it leads to neuron death. Deposits of amyloid β and aberrantly phosphorylated tau protein are known as pathological hallmarks of AD, but the underlying mechanisms have not yet been revealed. A high-throughput gene expression analysis previously showed that differentially expressed genes accompanying the progression of AD were more down-regulated than up-regulated in the later stages of AD. This suggested that the molecular networks and their constituent modules collapsed along with AD progression. In this study, by using gene expression profiles and protein interaction networks (PINs), we identified the PINs expressed in three brain regions: the entorhinal cortex (EC), hippocampus (HIP) and superior frontal gyrus (SFG). Dividing the expressed PINs into modules, we examined the stability of the modules with AD progression and with normal aging. We found that in the AD modules, the constituent proteins, interactions and cellular functions were not maintained between consecutive stages through all brain regions. Interestingly, the modules were collapsed with AD progression, specifically in the EC region. By identifying the modules that were affected by AD pathology, we found the transcriptional regulation-associated modules that interact with the proteasome-associated module via UCHL5 hub protein, which is a deubiquitinating enzyme. Considering PINs as a system made of network modules, we found that the modules relevant to the transcriptional regulation are disrupted in the EC region, which affects the ubiquitin-proteasome system. PMID:24348898

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

PubMed

Fujisawa, Takao; Filippakopoulos, Panagis

2017-04-01

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Photonic homeostatics

NASA Astrophysics Data System (ADS)

Liu, Timon C.; Li, Fan-Hui

2010-11-01

Photonic homeostatics is a discipline to study the establishment, maintenance, decay, upgrading and representation of function-specific homoestasis (FSH) by using photonics. FSH is a negative-feedback response of a biosystem to maintain the function-specific fluctuations inside the biosystem so that the function is perfectly performed. A stress may increase sirtuin 1 (SIRT1) activities above FSH-specific SIRT1 activity to induce a function far from its FSH. On the one hand, low level laser irradiation or monochromatic light (LLL) can not modulate a function in its FSH or a stress in its stress-specific homeostasis (StSH), but modulate a function far from its FSH or a stress far from its StSH. On the other hand, the biophotons from a biosystem with its function in its FSH should be less than the one from the biosystem with its function far from its FSH. The non-resonant interaction of low intensity laser irradiation or monochromatic light (LIL) and a kind of membrane protein can be amplified by all the membrane proteins if the function is far from its FSH. This amplification might hold for biophoton emission of the membrane protein so that the photonic spectroscopy can be used to represent the function far from its FSH, which is called photonomics.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

PubMed

Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

2017-07-16

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions.

PubMed

Won, Jung Hee; Park, Jung Sik; Ju, Hyun Hee; Kim, Soyeon; Suh-Kim, Haeyoung; Ghil, Sung Ho

2008-05-01

Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gbetagamma dimer; downstream effectors for its alpha subunit (Goalpha) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goalpha-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goalpha. Interactions between PLZF and Goalpha were confirmed using in vitro and in vivo affinity binding assays. Activated Goalpha interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/ialpha-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goalpha modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goalpha and its coupled receptor.

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

PubMed Central

Hu, Patrick J; Xu, Jinling; Ruvkun, Gary

2006-01-01

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues. PMID:16839187

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

The structure and function of Alzheimer's gamma secretase enzyme complex.

PubMed

Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

2009-01-01

The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

PubMed

Lambert, Matthias; Richard, Elodie; Duban-Deweer, Sophie; Krzewinski, Frederic; Deracinois, Barbara; Dupont, Erwan; Bastide, Bruno; Cieniewski-Bernard, Caroline

2016-09-01

The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation. Copyright © 2016 Elsevier B.V. All rights reserved.

A Conserved Deubiquitinating Enzyme Uses Intrinsically Disordered Regions to Scaffold Multiple Protein Interaction Sites*

PubMed Central

Reed, Benjamin J.; Locke, Melissa N.; Gardner, Richard G.

2015-01-01

In the canonical view of protein function, it is generally accepted that the three-dimensional structure of a protein determines its function. However, the past decade has seen a dramatic growth in the identification of proteins with extensive intrinsically disordered regions (IDRs), which are conformationally plastic and do not appear to adopt single three-dimensional structures. One current paradigm for IDR function is that disorder enables IDRs to adopt multiple conformations, expanding the ability of a protein to interact with a wide variety of disparate proteins. The capacity for many interactions is an important feature of proteins that occupy the hubs of protein networks, in particular protein-modifying enzymes that usually have a broad spectrum of substrates. One such protein modification is ubiquitination, where ubiquitin is attached to proteins through ubiquitin ligases (E3s) and removed through deubiquitinating enzymes. Numerous proteomic studies have found that thousands of proteins are dynamically regulated by cycles of ubiquitination and deubiquitination. Thus, how these enzymes target their wide array of substrates is of considerable importance for understanding the function of the cell's diverse ubiquitination networks. Here, we characterize a yeast deubiquitinating enzyme, Ubp10, that possesses IDRs flanking its catalytic protease domain. We show that Ubp10 possesses multiple, distinct binding modules within its IDRs that are necessary and sufficient for directing protein interactions important for Ubp10's known roles in gene silencing and ribosome biogenesis. The human homolog of Ubp10, USP36, also has IDRs flanking its catalytic domain, and these IDRs similarly contain binding modules important for protein interactions. This work highlights the significant protein interaction scaffolding abilities of IDRs in the regulation of dynamic protein ubiquitination. PMID:26149687

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin

PubMed Central

Rooijakkers, Bart J. M.

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain’s sequence–function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed. PMID:29782536

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

PubMed

Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway.

PubMed

Lin, Li; Hales, Chadwick M; Garber, Kathryn; Jin, Peng

2014-06-15

Polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. FTO is a nuclear protein and its physiological function remains largely unknown, but alterations in its expression in mice influence energy expenditure, food intake and, ultimately, body weight. To understand the molecular functions of FTO, we performed a yeast two-hybrid screen to identify the protein(s) that could directly interact with human FTO protein. Using multiple assays, we demonstrate that FTO interacts with three isoforms of calcium/calmodulin-dependent protein kinase II: α, β and γ, which are protein kinases that phosphorylate a broad range of substrates. This interaction is functional; overexpression of FTO delays the dephosphorylation of cAMP response element-binding protein (CREB) in human neuroblastoma (SK-N-SH) cells, which in turn leads to a dramatic increase in the expression of the CREB targets neuropeptide receptor 1 (NPY1R) and brain-derived neurotrophic factor (BDNF), which already are known to regulate food intake and energy homeostasis. Thus, our results suggest that FTO could modulate obesity by regulating the activity of the CREB signaling pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of the heat shock protein HSP27 on androgen receptor expression and function in prostate cancer cells.

PubMed

Stope, Matthias B; Schubert, Tina; Staar, Doreen; Rönnau, Cindy; Streitbörger, Andreas; Kroeger, Nils; Kubisch, Constanze; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

2012-06-01

Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.

Transcriptional Control by PARP-1: Chromatin Modulation, Enhancer-binding, Coregulation, and Insulation

PubMed Central

Kraus, W. Lee

2008-01-01

Summary The regulation of gene expression requires a wide array of protein factors that can modulate chromatin structure, act at enhancers, function as transcriptional coregulators, or regulate insulator function. Poly(ADP-ribose) polymerase-1 (PARP-1), an abundant and ubiquitous nuclear enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been implicated in all of these functions. Recent biochemical, genomic, proteomic, and cell-based studies have highlighted the role of PARP-1 in each of these processes and provided new insights about the molecular mechanisms governing PARP-1-dependent regulation of gene expression. In addition, these studies have demonstrated how PARP-1 functions as an integral part of cellular signaling pathways that culminate in gene regulatory outcomes. PMID:18450439

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

PubMed Central

Miller, Laurence J.

2010-01-01

It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures. PMID:20392808

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

The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

PubMed

Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

2007-09-01

Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

Endosomal protein traffic meets nuclear signal transduction head on.

PubMed

Horazdovsky, Bruce

2004-02-01

Rab5 plays a key role in controlling protein traffic through the early stages of the endocytic pathway. Previous studies on the modulators and effectors of Rab5 protein function have tied the regulation of several signal transduction pathways to the movement of protein through endocytic compartments. In the February 6, 2004, issue of Cell, Miaczynska et al. describe a surprising new link between Rab5 function and the nucleus by uncovering two new Rab5 effectors as potential regulators of the nucleosome remodeling and histone deacetylase protein complex NuRD/MeCP1.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

PubMed

Tenconi, Paula E; Giusto, Norma M; Salvador, Gabriela A; Mateos, Melina V

2016-12-01

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

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

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

PubMed

Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-10-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

PubMed Central

Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-01-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

The DAN family: modulators of TGF-β signaling and beyond.

PubMed

Nolan, Kristof; Thompson, Thomas B

2014-08-01

Extracellular binding proteins or antagonists are important factors that modulate ligands in the transforming growth factor (TGF-β) family. While the interplay between antagonists and ligands are essential for developmental and normal cellular processes, their imbalance can lead to the pathology of several disease states. In particular, recent studies have implicated members of the differential screening-selected gene in neuroblastoma (DAN) family in disease such as renal fibrosis, pulmonary arterial hypertension, and reactivation of metastatic cancer stem cells. DAN family members are known to inhibit the bone morphogenetic proteins (BMP) of the TGF-β family. However, unlike other TGF-β antagonist families, DAN family members have roles beyond ligand inhibition and can modulate Wnt and vascular endothelial growth factor (VEGF) signaling pathways. This review describes recent structural and functional advances that have expanded our understanding of DAN family proteins with regards to BMP inhibition and also highlights their emerging roles in the modulation of Wnt and VEGF signaling pathways. © 2014 The Protein Society.

The Biological Function of the Prion Protein: A Cell Surface Scaffold of Signaling Modules.

PubMed

Linden, Rafael

2017-01-01

The prion glycoprotein (PrP C ) is mostly located at the cell surface, tethered to the plasma membrane through a glycosyl-phosphatydil inositol (GPI) anchor. Misfolding of PrP C is associated with the transmissible spongiform encephalopathies (TSEs), whereas its normal conformer serves as a receptor for oligomers of the β-amyloid peptide, which play a major role in the pathogenesis of Alzheimer's Disease (AD). PrP C is highly expressed in both the nervous and immune systems, as well as in other organs, but its functions are controversial. Extensive experimental work disclosed multiple physiological roles of PrP C at the molecular, cellular and systemic levels, affecting the homeostasis of copper, neuroprotection, stem cell renewal and memory mechanisms, among others. Often each such process has been heralded as the bona fide function of PrP C , despite restricted attention paid to a selected phenotypic trait, associated with either modulation of gene expression or to the engagement of PrP C with a single ligand. In contrast, the GPI-anchored prion protein was shown to bind several extracellular and transmembrane ligands, which are required to endow that protein with the ability to play various roles in transmembrane signal transduction. In addition, differing sets of those ligands are available in cell type- and context-dependent scenarios. To account for such properties, we proposed that PrP C serves as a dynamic platform for the assembly of signaling modules at the cell surface, with widespread consequences for both physiology and behavior. The current review advances the hypothesis that the biological function of the prion protein is that of a cell surface scaffold protein, based on the striking similarities of its functional properties with those of scaffold proteins involved in the organization of intracellular signal transduction pathways. Those properties are: the ability to recruit spatially restricted sets of binding molecules involved in specific signaling; mediation of the crosstalk of signaling pathways; reciprocal allosteric regulation with binding partners; compartmentalized responses; dependence of signaling properties upon posttranslational modification; and stoichiometric requirements and/or oligomerization-dependent impact on signaling. The scaffold concept may contribute to novel approaches to the development of effective treatments to hitherto incurable neurodegenerative diseases, through informed modulation of prion protein-ligand interactions.

Protein Structure and Function: An Interdisciplinary Multimedia-Based Guided-Inquiry Education Module for the High School Science Classroom

ERIC Educational Resources Information Center

Bethel, Casey M.; Lieberman, Raquel L.

2014-01-01

Here we present a multidisciplinary educational unit intended for general, advanced placement, or international baccalaureate-level high school science, focused on the three-dimensional structure of proteins and their connection to function and disease. The lessons are designed within the framework of the Next Generation Science Standards to make…

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

PubMed Central

Delgado, M E; Dyck, L; Laussmann, M A; Rehm, M

2014-01-01

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death. PMID:24457955

NOVEL METHODS FOR TARGET PROTEIN IDENTIFICATION USING IMMUNOPRECIPITATION - LC/MS/MS

EPA Science Inventory

Proteomics provides a powerful approach to screen and analyze responses to environmental exposures which induce alterations in protein expression, phosphorylation. ubiquitinylation, oxidation. and modulation of general proteome function. Post-translational modifications (PTM) of ...

Metalloprotein entatic control of ligand-metal bonds quantified by ultrafast x-ray spectroscopy

DOE PAGES

Mara, Michael W.; Hadt, Ryan G.; Reinhard, Marco Eli; ...

2017-06-23

The multifunctional protein cytochrome c (cyt c) plays key roles in electron transport and apoptosis, switching function by modulating bonding between a heme iron and the sulfur in a methionine residue. This Fe–S(Met) bond is too weak to persist in the absence of protein constraints. We ruptured the bond in ferrous cyt c using an optical laser pulse and monitored the bond reformation within the protein active site using ultrafast x-ray pulses from an x-ray free-electron laser, determining that the Fe–S(Met) bond enthalpy is ~4 kcal/mol stronger than in the absence of protein constraints. As a result, the 4 kcal/molmore » is comparable with calculations of stabilization effects in other systems, demonstrating how biological systems use an entatic state for modest yet accessible energetics to modulate chemical function.« less

Assessing potential targets of calcium action in light-modulated gravitropism

NASA Technical Reports Server (NTRS)

Roux, S. J.

1995-01-01

Light, through the mediation of the pigment phytochrome, modulates the gravitropic response of the shoots and roots of many plants. The transduction of both light and gravity stimuli appears to involve Ca(2+)-regulated steps, one or more of which may represent points of intersection between the two transduction chains. To be confident that Ca2+ plays a critical role in stimulus-response coupling for gravitropism, it will be important to identify specific targets of Ca2+ action whose function can be clearly linked to the regulation of growth. Calcium typically exerts its influence on cell metabolism through binding to and activating key regulatory proteins. The three best characterized of these proteins in plants are the calmodulins, calcium-dependent protein kinases, and annexins. In this review we summarize what is known about the structure and function of these proteins and speculate on how their activation by Ca2+ could influence the differential growth response of gravitropism.

Dietary Nutrients and Bioactive Substances Modulate Heat Shock Protein (HSP) Expression: A Review.

PubMed

Moura, Carolina Soares; Lollo, Pablo Christiano Barboza; Morato, Priscila Neder; Amaya-Farfan, Jaime

2018-05-28

Interest in the heat shock proteins (HSPs), as a natural physiological toolkit of living organisms, has ranged from their chaperone function in nascent proteins to the remedial role following cell stress. As part of the defence system, HSPs guarantee cell tolerance against a variety of stressors, including exercise, oxidative stress, hyper and hypothermia, hyper and hypoxia and improper diets. For the past couple of decades, research on functional foods has revealed a number of substances likely to trigger cell protection through mechanisms that involve the induction of HSP expression. This review will summarize the occurrence of the most easily inducible HSPs and describe the effects of dietary proteins, peptides, amino acids, probiotics, high-fat diets and other food-derived substances reported to induce HSP response in animals and humans studies. Future research may clarify the mechanisms and explore the usefulness of this natural alternative of defense and the modulating mechanism of each substance.

Metalloprotein entatic control of ligand-metal bonds quantified by ultrafast x-ray spectroscopy

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

Mara, Michael W.; Hadt, Ryan G.; Reinhard, Marco Eli

The multifunctional protein cytochrome c (cyt c) plays key roles in electron transport and apoptosis, switching function by modulating bonding between a heme iron and the sulfur in a methionine residue. This Fe–S(Met) bond is too weak to persist in the absence of protein constraints. We ruptured the bond in ferrous cyt c using an optical laser pulse and monitored the bond reformation within the protein active site using ultrafast x-ray pulses from an x-ray free-electron laser, determining that the Fe–S(Met) bond enthalpy is ~4 kcal/mol stronger than in the absence of protein constraints. As a result, the 4 kcal/molmore » is comparable with calculations of stabilization effects in other systems, demonstrating how biological systems use an entatic state for modest yet accessible energetics to modulate chemical function.« less

Prior knowledge guided active modules identification: an integrated multi-objective approach.

PubMed

Chen, Weiqi; Liu, Jing; He, Shan

2017-03-14

Active module, defined as an area in biological network that shows striking changes in molecular activity or phenotypic signatures, is important to reveal dynamic and process-specific information that is correlated with cellular or disease states. A prior information guided active module identification approach is proposed to detect modules that are both active and enriched by prior knowledge. We formulate the active module identification problem as a multi-objective optimisation problem, which consists two conflicting objective functions of maximising the coverage of known biological pathways and the activity of the active module simultaneously. Network is constructed from protein-protein interaction database. A beta-uniform-mixture model is used to estimate the distribution of p-values and generate scores for activity measurement from microarray data. A multi-objective evolutionary algorithm is used to search for Pareto optimal solutions. We also incorporate a novel constraints based on algebraic connectivity to ensure the connectedness of the identified active modules. Application of proposed algorithm on a small yeast molecular network shows that it can identify modules with high activities and with more cross-talk nodes between related functional groups. The Pareto solutions generated by the algorithm provides solutions with different trade-off between prior knowledge and novel information from data. The approach is then applied on microarray data from diclofenac-treated yeast cells to build network and identify modules to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Gene ontology analysis is applied to the identified modules for biological interpretation. Integrating knowledge of functional groups into the identification of active module is an effective method and provides a flexible control of balance between pure data-driven method and prior information guidance.

Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

DOE PAGES

McMillan, Janet R.; Brodin, Jeffrey D.; Millan, Jaime A.; ...

2017-01-25

Here, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using Beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When assembled into crystalline superlattices with AuNPs, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB 2 packing. We probe the role of protein oligonucleotide number and conjugatemore » size on this observation, which revealed the importance of oligonucleotide distribution and number in this observed assembly behavior. These results indicate that proteins with defined DNA-modification patterns are powerful tools to control the nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.« less

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

An ultra-HTS process for the identification of small molecule modulators of orphan G-protein-coupled receptors.

PubMed

Cacace, Angela; Banks, Martyn; Spicer, Timothy; Civoli, Francesca; Watson, John

2003-09-01

G-protein-coupled receptors (GPCRs) are the most successful target proteins for drug discovery research to date. More than 150 orphan GPCRs of potential therapeutic interest have been identified for which no activating ligands or biological functions are known. One of the greatest challenges in the pharmaceutical industry is to link these orphan GPCRs with human diseases. Highly automated parallel approaches that integrate ultra-high throughput and focused screening can be used to identify small molecule modulators of orphan GPCRs. These small molecules can then be employed as pharmacological tools to explore the function of orphan receptors in models of human disease. In this review, we describe methods that utilize powerful ultra-high-throughput screening technologies to identify surrogate ligands of orphan GPCRs.

Identification of functional modules using network topology and high-throughput data.

PubMed

Ulitsky, Igor; Shamir, Ron

2007-01-26

With the advent of systems biology, biological knowledge is often represented today by networks. These include regulatory and metabolic networks, protein-protein interaction networks, and many others. At the same time, high-throughput genomics and proteomics techniques generate very large data sets, which require sophisticated computational analysis. Usually, separate and different analysis methodologies are applied to each of the two data types. An integrated investigation of network and high-throughput information together can improve the quality of the analysis by accounting simultaneously for topological network properties alongside intrinsic features of the high-throughput data. We describe a novel algorithmic framework for this challenge. We first transform the high-throughput data into similarity values, (e.g., by computing pairwise similarity of gene expression patterns from microarray data). Then, given a network of genes or proteins and similarity values between some of them, we seek connected sub-networks (or modules) that manifest high similarity. We develop algorithms for this problem and evaluate their performance on the osmotic shock response network in S. cerevisiae and on the human cell cycle network. We demonstrate that focused, biologically meaningful and relevant functional modules are obtained. In comparison with extant algorithms, our approach has higher sensitivity and higher specificity. We have demonstrated that our method can accurately identify functional modules. Hence, it carries the promise to be highly useful in analysis of high throughput data.

Modulation of protein stability and aggregation properties by surface charge engineering.

PubMed

Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

2013-09-01

An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

CNG-Modulin: a novel Ca-dependent modulator of ligand sensitivity in cone photoreceptor cGMP-gated ion channels

PubMed Central

Rebrik, Tatiana I.; Botchkina, Inna; Arshavsky, Vadim Y.; Craft, Cheryl M.; Korenbrot, Juan I.

2012-01-01

The transduction current in several different types of sensory neurons arises from the activity of cyclic nucleotide gated ion channels (CNG channels). The channels in these sensory neurons vary in structure and function, yet each one demonstrates calcium-dependent modulation of ligand sensitivity mediated by the interaction of the channel with a soluble modulator protein. In cone photoreceptors, the molecular identity of the modulator protein was previously unknown. We report the discovery and characterization of CNG-modulin, a novel 301 amino acid protein that interacts with the N-terminus of the β-subunit of the cGMP-gated channel, and modulates the cGMP sensitivity of the channels in cone photoreceptors of striped bass (Morone saxitilis). Immunohistochemistry and single cell PCR demonstrate that CNG-modulin is expressed in cone, but not rod photoreceptors. Adding purified recombinant CNG-modulin to cone membrane patches containing the native CNG channels shifts the midpoint of cGMP-dependence from ~91 μM in the absence of Ca2+ to ~332 μM in the presence of 20 μM Ca2+. At a fixed cGMP concentration, the midpoint of the Ca2+ dependence is ~857 nM Ca2+. These restored physiological features are statistically indistinguishable from the effects of the endogenous modulator. CNG-modulin binds Ca2+ with a concentration dependence that matches the calcium dependence of channel modulation. We conclude that CNG-modulin is the authentic Ca2+-dependent modulator of cone CNG channel ligand sensitivity. CNG-modulin is expressed in other tissues, such as brain, olfactory epithelium and the inner ear and may modulate the function of ion channels in those tissues as well. PMID:22378887

CNG-modulin: a novel Ca-dependent modulator of ligand sensitivity in cone photoreceptor cGMP-gated ion channels.

PubMed

Rebrik, Tatiana I; Botchkina, Inna; Arshavsky, Vadim Y; Craft, Cheryl M; Korenbrot, Juan I

2012-02-29

The transduction current in several different types of sensory neurons arises from the activity of cyclic nucleotide-gated (CNG) ion channels. The channels in these sensory neurons vary in structure and function, yet each one demonstrates calcium-dependent modulation of ligand sensitivity mediated by the interaction of the channel with a soluble modulator protein. In cone photoreceptors, the molecular identity of the modulator protein was previously unknown. We report the discovery and characterization of CNG-modulin, a novel 301 aa protein that interacts with the N terminus of the β subunit of the cGMP-gated channel and modulates the cGMP sensitivity of the channels in cone photoreceptors of striped bass (Morone saxatilis). Immunohistochemistry and single-cell PCR demonstrate that CNG-modulin is expressed in cone but not rod photoreceptors. Adding purified recombinant CNG-modulin to cone membrane patches containing the native CNG channels shifts the midpoint of cGMP dependence from ∼91 μM in the absence of Ca(2+) to ∼332 μM in the presence of 20 μM Ca(2+). At a fixed cGMP concentration, the midpoint of the Ca(2+) dependence is ∼857 nM Ca(2+). These restored physiological features are statistically indistinguishable from the effects of the endogenous modulator. CNG-modulin binds Ca(2+) with a concentration dependence that matches the calcium dependence of channel modulation. We conclude that CNG-modulin is the authentic Ca(2+)-dependent modulator of cone CNG channel ligand sensitivity. CNG-modulin is expressed in other tissues, such as brain, olfactory epithelium, and the inner ear, and may modulate the function of ion channels in those tissues as well.

MRF Family Genes Are Involved in Translation Control, Especially under Energy-Deficient Conditions, and Their Expression and Functions Are Modulated by the TOR Signaling Pathway[OPEN

PubMed Central

Lee, Du-Hwa; Park, Seung Jun; Ahn, Chang Sook

2017-01-01

Dynamic control of protein translation in response to the environment is essential for the survival of plant cells. Target of rapamycin (TOR) coordinates protein synthesis with cellular energy/nutrient availability through transcriptional modulation and phosphorylation of the translation machinery. However, mechanisms of TOR-mediated translation control are poorly understood in plants. Here, we report that Arabidopsis thaliana MRF (MA3 DOMAIN-CONTAINING TRANSLATION REGULATORY FACTOR) family genes encode translation regulatory factors under TOR control, and their functions are particularly important in energy-deficient conditions. Four MRF family genes (MRF1-MRF4) are transcriptionally induced by dark and starvation (DS). Silencing of multiple MRFs increases susceptibility to DS and treatment with a TOR inhibitor, while MRF1 overexpression decreases susceptibility. MRF proteins interact with eIF4A and cofractionate with ribosomes. MRF silencing decreases translation activity, while MRF1 overexpression increases it, accompanied by altered ribosome patterns, particularly in DS. Furthermore, MRF deficiency in DS causes altered distribution of mRNAs in sucrose gradient fractions and accelerates rRNA degradation. MRF1 is phosphorylated in vivo and phosphorylated by S6 kinases in vitro. MRF expression and MRF1 ribosome association and phosphorylation are modulated by cellular energy status and TOR activity. We discuss possible mechanisms of the function of MRF family proteins under normal and energy-deficient conditions and their functional link with the TOR pathway. PMID:29084871

Frodo proteins: modulators of Wnt signaling in vertebrate development.

PubMed

Brott, Barbara K; Sokol, Sergei Y

2005-09-01

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.

Engineered TAL Effector modulators for the large-scale gain-of-function screening

PubMed Central

Zhang, Hanshuo; Li, Juan; Hou, Sha; Wang, Gancheng; Jiang, Mingjun; Sun, Changhong; Hu, Xiongbing; Zhuang, Fengfeng; Dai, Zhifei; Dai, Junbiao; Xi, Jianzhong Jeff

2014-01-01

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE–DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE–TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation. PMID:24939900

Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

PubMed

Chaurasia, Satya Prakash; Deswal, Renu

2017-02-01

The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H 2 O 2 ) induced changes in the gene expression have been reported, but efforts to detect H 2 O 2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H 2 O 2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

PubMed

Pan, Joshua; Meyers, Robin M; Michel, Brittany C; Mashtalir, Nazar; Sizemore, Ann E; Wells, Jonathan N; Cassel, Seth H; Vazquez, Francisca; Weir, Barbara A; Hahn, William C; Marsh, Joseph A; Tsherniak, Aviad; Kadoch, Cigall

2018-05-23

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Long non-coding RNAs and mRNAs profiling during spleen development in pig.

PubMed

Che, Tiandong; Li, Diyan; Jin, Long; Fu, Yuhua; Liu, Yingkai; Liu, Pengliang; Wang, Yixin; Tang, Qianzi; Ma, Jideng; Wang, Xun; Jiang, Anan; Li, Xuewei; Li, Mingzhou

2018-01-01

Genome-wide transcriptomic studies in humans and mice have become extensive and mature. However, a comprehensive and systematic understanding of protein-coding genes and long non-coding RNAs (lncRNAs) expressed during pig spleen development has not been achieved. LncRNAs are known to participate in regulatory networks for an array of biological processes. Here, we constructed 18 RNA libraries from developing fetal pig spleen (55 days before birth), postnatal pig spleens (0, 30, 180 days and 2 years after birth), and the samples from the 2-year-old Wild Boar. A total of 15,040 lncRNA transcripts were identified among these samples. We found that the temporal expression pattern of lncRNAs was more restricted than observed for protein-coding genes. Time-series analysis showed two large modules for protein-coding genes and lncRNAs. The up-regulated module was enriched for genes related to immune and inflammatory function, while the down-regulated module was enriched for cell proliferation processes such as cell division and DNA replication. Co-expression networks indicated the functional relatedness between protein-coding genes and lncRNAs, which were enriched for similar functions over the series of time points examined. We identified numerous differentially expressed protein-coding genes and lncRNAs in all five developmental stages. Notably, ceruloplasmin precursor (CP), a protein-coding gene participating in antioxidant and iron transport processes, was differentially expressed in all stages. This study provides the first catalog of the developing pig spleen, and contributes to a fuller understanding of the molecular mechanisms underpinning mammalian spleen development.

Insulin Action in Brain Regulates Systemic Metabolism and Brain Function

PubMed Central

Kleinridders, André; Ferris, Heather A.; Cai, Weikang

2014-01-01

Insulin receptors, as well as IGF-1 receptors and their postreceptor signaling partners, are distributed throughout the brain. Insulin acts on these receptors to modulate peripheral metabolism, including regulation of appetite, reproductive function, body temperature, white fat mass, hepatic glucose output, and response to hypoglycemia. Insulin signaling also modulates neurotransmitter channel activity, brain cholesterol synthesis, and mitochondrial function. Disruption of insulin action in the brain leads to impairment of neuronal function and synaptogenesis. In addition, insulin signaling modulates phosphorylation of tau protein, an early component in the development of Alzheimer disease. Thus, alterations in insulin action in the brain can contribute to metabolic syndrome, and the development of mood disorders and neurodegenerative diseases. PMID:24931034

Moonlighting adenosine deaminase: a target protein for drug development.

PubMed

Cortés, Antoni; Gracia, Eduard; Moreno, Estefania; Mallol, Josefa; Lluís, Carme; Canela, Enric I; Casadó, Vicent

2015-01-01

Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest. © 2014 Wiley Periodicals, Inc.

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease

PubMed Central

Kwon, Michelle; Pavlov, Tengis S.; Nozu, Kandai; Rasmussen, Shauna A.; Ilatovskaya, Daria V.; Lerch-Gaggl, Alexandra; North, Lauren M.; Kim, Hyunho; Qian, Feng; Sweeney, William E.; Avner, Ellis D.; Blumer, Joe B.; Staruschenko, Alexander; Park, Frank

2012-01-01

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1+/+ and Gpsm1+/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease. PMID:23236168

Docking, thermodynamics and molecular dynamics (MD) studies of a non-canonical protease inhibitor, MP-4, from Mucuna pruriens.

PubMed

Kumar, Ashish; Kaur, Harmeet; Jain, Abha; Nair, Deepak T; Salunke, Dinakar M

2018-01-12

Sequence and structural homology suggests that MP-4 protein from Mucuna pruriens belongs to Kunitz-type protease inhibitor family. However, biochemical assays showed that this protein is a poor inhibitor of trypsin. To understand the basis of observed poor inhibition, thermodynamics and molecular dynamics (MD) simulation studies on binding of MP-4 to trypsin were carried out. Molecular dynamics simulations revealed that temperature influences the spectrum of conformations adopted by the loop regions in the MP-4 structure. At an optimal temperature, MP-4 achieves maximal binding while above and below the optimum temperature, its functional activity is hampered due to unfavourable flexibility and relative rigidity, respectively. The low activity at normal temperature is due to the widening of the conformational spectrum of the Reactive Site Loop (RSL) that reduces the probability of formation of stabilizing contacts with trypsin. The unique sequence of the RSL enhances flexibility at ambient temperature and thus reduces its ability to inhibit trypsin. This study shows that temperature influences the function of a protein through modulation in the structure of functional domain of the protein. Modulation of function through appearance of new sequences that are more sensitive to temperature may be a general strategy for evolution of new proteins.

Functionalization of alkyne-terminated thermally hydrocarbonized porous silicon nanoparticles with targeting peptides and antifouling polymers: effect on the human plasma protein adsorption.

PubMed

Wang, Chang-Fang; Mäkilä, Ermei M; Bonduelle, Colin; Rytkönen, Jussi; Raula, Janne; Almeida, Sérgio; Närvänen, Ale; Salonen, Jarno J; Lecommandoux, Sebastien; Hirvonen, Jouni T; Santos, Hélder A

2015-01-28

Porous silicon (PSi) nanomaterials combine a high drug loading capacity and tunable surface chemistry with various surface modifications to meet the requirements for biomedical applications. In this work, alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) nanoparticles were fabricated and postmodified using five bioactive molecules (targeting peptides and antifouling polymers) via a single-step click chemistry to modulate the bioactivity of the THCPSi nanoparticles, such as enhancing the cellular uptake and reducing the plasma protein association. The size of the nanoparticles after modification was increased from 176 to 180-220 nm. Dextran 40 kDa modified THCPSi nanoparticles showed the highest stability in aqueous buffer. Both peptide- and polymer-functionalized THCPSi nanoparticles showed an extensive cellular uptake which was dependent on the functionalized moieties presented on the surface of the nanoparticles. The plasma protein adsorption study showed that the surface modification with different peptides or polymers induced different protein association profiles. Dextran 40 kDa functionalized THCPSi nanoparticles presented the least protein association. Overall, these results demonstrate that the "click" conjugation of the biomolecules onto the alkyne-terminated THCPSi nanoparticles is a versatile and simple approach to modulate the surface chemistry, which has high potential for biomedical applications.

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Splice-mediated Variants of Proteins (SpliVaP) - data and characterization of changes in signatures among protein isoforms due to alternative splicing.

PubMed

Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

2008-10-02

It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. (i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. (i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms - since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii) The reported resource (at http://www.bioinformatica.crs4.org/tools/dbs/splivap/) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures.

Extensions of PDZ domains as important structural and functional elements.

PubMed

Wang, Conan K; Pan, Lifeng; Chen, Jia; Zhang, Mingjie

2010-08-01

'Divide and conquer' has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions 'extensions'. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Using bioinformatics, we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results ( http://bcz102.ust.hk/pdzex/ ). We propose, using well-studied PDZ domains as illustrative examples, that the roles of PDZ extensions can be classified into at least four categories: 1) protein dynamics-based modulation of target binding affinity, 2) provision of binding sites for macro-molecular assembly, 3) structural integration of multi-domain modules, and 4) expansion of the target ligand-binding pocket. Our review highlights the potential structural and functional importance of domain extensions, highlighting the significance of looking beyond the canonical boundaries of protein domains in general.

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

PubMed Central

Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

2014-01-01

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

PubMed

Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

2009-10-01

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Handling gene and protein names in the age of bioinformatics: the special challenge of secreted multimodular bacterial enzymes such as the cbhA/cbh9A gene of Clostridium thermocellum.

PubMed

Schwarz, Wolfgang H; Brunecky, Roman; Broeker, Jannis; Liebl, Wolfgang; Zverlov, Vladimir V

2018-02-26

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with a respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.

Carboxysomes: metabolic modules for CO 2 fixation

DOE PAGES

Turmo, Aiko; Gonzalez-Esquer, Cesar Raul; Kerfeld, Cheryl A.

2017-08-14

The carboxysome is a bacterial microcompartment encapsulating the enzymes carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase. As the site of CO 2 fixation, it serves an essential role in the carbon dioxide concentrating mechanism of many chemoautotrophs and all cyanobacteria. Carboxysomes and other bacterial microcompartments self-assemble through specific protein–protein interactions that are typically mediated by conserved protein domains. In this review, we frame our current understanding of carboxysomes in the context of their component protein domains with their inherent function as the ‘building blocks’ of carboxysomes. These building blocks are organized in genetic modules (conserved chromosomal loci) that encode for carboxysomes andmore » ancillary proteins essential for the integration of the organelle with the rest of cellular metabolism. This conceptual framework provides the foundation for ‘plug-and-play’ engineering of carboxysomes as CO 2 fixation modules in a variety of biotechnological applications.« less

Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

PubMed

Bahr, Ben A; Wisniewski, Meagan L; Butler, David

2012-04-01

Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance protein clearance, promote synaptic integrity, and slow the progression of dementia.

Rubisco Activase Activity Assays

USDA-ARS?s Scientific Manuscript database

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, Rubisco. This action modulates the activation state of Rubisco by removing tightly-bound inhibitory s...

The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.

PubMed

Jacobsen, Annette V; Murphy, James M

2017-06-15

Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic diversity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

J domain independent functions of J proteins.

PubMed

Ajit Tamadaddi, Chetana; Sahi, Chandan

2016-07-01

Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

Lipid nanotechnologies for structural studies of membrane-associated proteins.

PubMed

Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

2014-11-01

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

Current opinion in Microbiology Roles of adaptor proteins in regulation of bacterial proteolysis

PubMed Central

Battesti, Aurelia; Gottesman, Susan

2013-01-01

Elimination of non-functional or unwanted proteins is critical for cell growth and regulation. In bacteria, ATP-dependent proteases target cytoplasmic proteins for degradation, contributing to both protein quality control and regulation of specific proteins, thus playing roles parallel to that of the proteasome in eukaryotic cells. Adaptor proteins provide a way to modulate the substrate specificity of the proteases and allow regulated proteolysis. Advances over the past few years have provided new insight into how adaptor proteins interact with both substrates and proteases and how adaptor functions are regulated. An important advance has come with the recognition of the critical roles of anti-adaptor proteins in regulating adaptor availability. PMID:23375660

Deciphering the BAR code of membrane modulators.

PubMed

Salzer, Ulrich; Kostan, Julius; Djinović-Carugo, Kristina

2017-07-01

The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.

HIV Nef-mediated cellular phenotypes are differentially expressed as a function of intracellular Nef concentrations.

PubMed

Liu, X; Schrager, J A; Lange, G D; Marsh, J W

2001-08-31

Nef is a regulatory protein encoded by the genome of both human and simian immunodeficiency virus. Its expression in T cells leads to CD4 and major histocompatibility complex class I modulation and either enhancement or suppression of T cell activation. How this viral protein achieves multiple and at times opposing activities has been unclear. Through direct measurements of Nef and the Nef-GFP fusion protein, we find that these events are mediated by different Nef concentrations. Relative to the intracellular concentration that down-modulates surface CD4, an order of magnitude increase in Nef-GFP expression is required for a comparable modulation of major histocompatibility complex class I, and a further 3-fold increase is necessary to suppress T cell activation.

CRISPR adaptive immune systems of Archaea

PubMed Central

Vestergaard, Gisle; Garrett, Roger A; Shah, Shiraz A

2014-01-01

CRISPR adaptive immune systems were analyzed for all available completed genomes of archaea, which included representatives of each of the main archaeal phyla. Initially, all proteins encoded within, and proximal to, CRISPR-cas loci were clustered and analyzed using a profile–profile approach. Then cas genes were assigned to gene cassettes and to functional modules for adaptation and interference. CRISPR systems were then classified primarily on the basis of their concatenated Cas protein sequences and gene synteny of the interference modules. With few exceptions, they could be assigned to the universal Type I or Type III systems. For Type I, subtypes I-A, I-B, and I-D dominate but the data support the division of subtype I-B into two subtypes, designated I-B and I-G. About 70% of the Type III systems fall into the universal subtypes III-A and III-B but the remainder, some of which are phyla-specific, diverge significantly in Cas protein sequences, and/or gene synteny, and they are classified separately. Furthermore, a few CRISPR systems that could not be assigned to Type I or Type III are categorized as variant systems. Criteria are presented for assigning newly sequenced archaeal CRISPR systems to the different subtypes. Several accessory proteins were identified that show a specific gene linkage, especially to Type III interference modules, and these may be cofunctional with the CRISPR systems. Evidence is presented for extensive exchange having occurred between adaptation and interference modules of different archaeal CRISPR systems, indicating the wide compatibility of the functionally diverse interference complexes with the relatively conserved adaptation modules. PMID:24531374

Adipokines in Liver Cirrhosis.

PubMed

Buechler, Christa; Haberl, Elisabeth M; Rein-Fischboeck, Lisa; Aslanidis, Charalampos

2017-06-29

Liver fibrosis can progress to cirrhosis, which is considered a serious disease. The Child-Pugh score and the model of end-stage liver disease score have been established to assess residual liver function in patients with liver cirrhosis. The development of portal hypertension contributes to ascites, variceal bleeding and further complications in these patients. A transjugular intrahepatic portosystemic shunt (TIPS) is used to lower portal pressure, which represents a major improvement in the treatment of patients. Adipokines are proteins released from adipose tissue and modulate hepatic fibrogenesis. These proteins affect various biological processes that are involved in liver function, including angiogenesis, vasodilation, inflammation and deposition of extracellular matrix proteins. The best studied adipokines are adiponectin and leptin. Adiponectin protects against hepatic inflammation and fibrogenesis, and leptin functions as a profibrogenic factor. These and other adipokines are supposed to modulate disease severity in patients with liver cirrhosis. Consequently, circulating levels of these proteins have been analyzed to identify associations with parameters of hepatic function, portal hypertension and its associated complications in patients with liver cirrhosis. This review article briefly addresses the role of adipokines in hepatitis and liver fibrosis. Here, studies having analyzed these proteins in systemic blood in cirrhotic patients are listed to identify adipokines that are comparably changed in the different cohorts of patients with liver cirrhosis. Some studies measured these proteins in systemic, hepatic and portal vein blood or after TIPS to specify the tissues contributing to circulating levels of these proteins and the effect of portal hypertension, respectively.

Adipokines in Liver Cirrhosis

PubMed Central

Haberl, Elisabeth M.; Rein-Fischboeck, Lisa; Aslanidis, Charalampos

2017-01-01

Liver fibrosis can progress to cirrhosis, which is considered a serious disease. The Child-Pugh score and the model of end-stage liver disease score have been established to assess residual liver function in patients with liver cirrhosis. The development of portal hypertension contributes to ascites, variceal bleeding and further complications in these patients. A transjugular intrahepatic portosystemic shunt (TIPS) is used to lower portal pressure, which represents a major improvement in the treatment of patients. Adipokines are proteins released from adipose tissue and modulate hepatic fibrogenesis. These proteins affect various biological processes that are involved in liver function, including angiogenesis, vasodilation, inflammation and deposition of extracellular matrix proteins. The best studied adipokines are adiponectin and leptin. Adiponectin protects against hepatic inflammation and fibrogenesis, and leptin functions as a profibrogenic factor. These and other adipokines are supposed to modulate disease severity in patients with liver cirrhosis. Consequently, circulating levels of these proteins have been analyzed to identify associations with parameters of hepatic function, portal hypertension and its associated complications in patients with liver cirrhosis. This review article briefly addresses the role of adipokines in hepatitis and liver fibrosis. Here, studies having analyzed these proteins in systemic blood in cirrhotic patients are listed to identify adipokines that are comparably changed in the different cohorts of patients with liver cirrhosis. Some studies measured these proteins in systemic, hepatic and portal vein blood or after TIPS to specify the tissues contributing to circulating levels of these proteins and the effect of portal hypertension, respectively. PMID:28661458

Selective Chemical Modulation of Gene Transcription Favors Oligodendrocyte Lineage Progression

PubMed Central

Plotnikov, Alexander N.; Zhang, Guangtao; Zeng, Lei; Kaur, Jasbir; Moy, Gregory; Rusinova, Elena; Rodriguez, Yoel; Matikainen, Bridget; Vincek, Adam; Joshua, Jennifer; Casaccia, Patrizia; Zhou, Ming-Ming

2014-01-01

SUMMARY Lysine acetylation regulates gene expression through modulating protein-protein interactions in chromatin. Chemical inhibition of acetyl-lysine binding bromodomains of the major chromatin regulators BET (bromodomain and extra-terminal domain) proteins, has been shown to effectively block cell proliferation in cancer and inflammation. However, whether selective inhibition of individual BET bromodomains has distinctive functional consequences, remains only partially understood. In this study, we show that selective chemical inhibition of the first bromodomain of BET proteins using our newly designed small molecule inhibitor, Olinone, accelerated the progression of mouse primary oligodendrocyte progenitors towards differentiation, while inhibition of both bromodomains of BET proteins hindered differentiation. This effect was target-specific, as it was not detected in cells treated with inactive analogues and independent of any effect on proliferation. Therefore, selective chemical modulation of individual bromodomains, rather than use of broad-based inhibitors may enhance regenerative strategies in disorders characterized by myelin loss such as aging and neurodegeneration. PMID:24954007

Functions of intrinsic disorder in transmembrane proteins.

PubMed

Kjaergaard, Magnus; Kragelund, Birthe B

2017-09-01

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.

Arabidopsis Protein Phosphatase DBP1 Nucleates a Protein Network with a Role in Regulating Plant Defense

PubMed Central

Naumann, Kai; Lassowskat, Ines; Navarrete-Gómez, Marisa; Scheel, Dierk; Vera, Pablo

2014-01-01

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis. PMID:24595057

Protein Conformational Populations and Functionally Relevant Sub-states

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

Agarwal, Pratul K; Burger, Virginia; Savol, Andrej

2013-01-01

Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of themore » protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow it to attain the transition state, therefore promoting the reaction mechanism. In the long term, this emerging view of proteins with conformational substates has broad implications for improving our understanding of enzymes, enzyme engineering, and better drug design. Researchers have already used photoactivation to modulate protein conformations as a strategy to develop a hypercatalytic enzyme. In addition, the alteration of the conformational substates through binding of ligands at locations other than the active site provides the basis for the design of new medicines through allosteric modulation.« less

Dual Coordination of Post Translational Modifications in Human Protein Networks

PubMed Central

Woodsmith, Jonathan; Kamburov, Atanas; Stelzl, Ulrich

2013-01-01

Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling. PMID:23505349

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

PubMed Central

Barik, Sailen

2008-01-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the α-helices and β-strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the α-helix and the β-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures. PMID:15381847

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture.

PubMed

Barik, Sailen

2004-09-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the alpha-helices and beta-strands of proteins than within the more flexible linker regions ('turns' and 'loops') connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the alpha-helix and the beta-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

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

PubMed Central

2014-01-01

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

Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

PubMed Central

Samrat, Subodh Kumar; Li, Wen; Singh, Shakti; Kumar, Rakesh; Agrawal, Babita

2014-01-01

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans. PMID:24475147

Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development.

PubMed

Cho, Young-Hee; Hong, Jung-Woo; Kim, Eun-Chul; Yoo, Sang-Dong

2012-04-01

Sucrose-nonfermentation1-related protein kinase1 (SnRK1) is an evolutionarily conserved energy sensor protein that regulates gene expression in response to energy depletion in plants. Efforts to elucidate the functions and mechanisms of this protein kinase are hampered, however, by inherent growth defects of snrk1-null mutant plants. To overcome these limitations and study SnRK1 functions in vivo, we applied a method combining transient expression in leaf mesophyll protoplasts and stable expression in transgenic plants. We found that both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) SnRK1 activities critically influence stress-inducible gene expression and the induction of stress tolerance. Genetic, molecular, and chromatin immunoprecipitation analyses further revealed that the nuclear SnRK1 modulated target gene transcription in a submergence-dependent manner. From early seedling development through late senescence, SnRK1 activities appeared to modulate developmental processes in the plants. Our findings offer insight into the regulatory functions of plant SnRK1 in stress-responsive gene regulation and in plant growth and development throughout the life cycle.

DYNLL/LC8 Protein Controls Signal Transduction through the Nek9/Nek6 Signaling Module by Regulating Nek6 Binding to Nek9

PubMed Central

Regué, Laura; Sdelci, Sara; Bertran, M. Teresa; Caelles, Carme; Reverter, David; Roig, Joan

2011-01-01

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser944, a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation. PMID:21454704

Controlling adsorption and passivation properties of bovine serum albumin on silica surfaces by ionic strength modulation and cross-linking.

PubMed

Park, Jae Hyeon; Sut, Tun Naw; Jackman, Joshua A; Ferhan, Abdul Rahim; Yoon, Bo Kyeong; Cho, Nam-Joon

2017-03-29

Understanding the physicochemical factors that influence protein adsorption onto solid supports holds wide relevance for fundamental insights into protein structure and function as well as for applications such as surface passivation. Ionic strength is a key parameter that influences protein adsorption, although how its modulation might be utilized to prepare well-coated protein adlayers remains to be explored. Herein, we investigated how ionic strength can be utilized to control the adsorption and passivation properties of bovine serum albumin (BSA) on silica surfaces. As protein stability in solution can influence adsorption kinetics, the size distribution and secondary structure of proteins in solution were first characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and circular dichroism (CD) spectroscopy. A non-monotonic correlation between ionic strength and protein aggregation was observed and attributed to colloidal agglomeration, while the primarily α-helical character of the protein in solution was maintained in all cases. Quartz crystal microbalance-dissipation (QCM-D) experiments were then conducted in order to track protein adsorption onto silica surfaces as a function of ionic strength, and the measurement responses indicated that total protein uptake at saturation coverage is lower with increasing ionic strength. In turn, the QCM-D data and the corresponding Voigt-Voinova model analysis support that the surface area per bound protein molecule is greater with increasing ionic strength. While higher protein uptake under lower ionic strengths by itself did not result in greater surface passivation under subsequent physiologically relevant conditions, the treatment of adsorbed protein layers with a gluteraldehyde cross-linking agent stabilized the bound protein in this case and significantly improved surface passivation. Collectively, our findings demonstrate that ionic strength modulation influences BSA adsorption uptake on account of protein spreading and can be utilized in conjunction with covalent cross-linking strategies to prepare well-coated protein adlayers for improved surface passivation.

Elucidating the structure and function of S100 proteins in membranes

NASA Astrophysics Data System (ADS)

Valenzuela, Stella M.; Berkahn, Mark; Martin, Donald K.; Huynh, Thuan; Yang, Zheng; Geczy, Carolyn L.

2006-01-01

S100 proteins are important Ca 2+-binding proteins involved in vital cellular functions including the modulation of cell growth, migration and differentiation, regulation of intracellular signal transduction/phosphorylation pathways, energy metabolism, cytoskeletal interactions and modulation of ion channels. Furthermore, they are implicated in oncogenesis and numerous other disease states. Three S100 proteins: S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and monocytes. At low levels of intracellular Ca 2+, S100A8 and S100A9 are located predominantly in the cytosol but when Ca 2+ concentrations are elevated as a consequence of activation, they translocate to membranes and complex with cytoskeletal components such as vimentin. The functions of S100A8 and S100A9 at the plasma membrane remain unclear. A possible role may be the regulation of ion channel proteins. The current study uses the techniques of Atomic Force Microscopy and production of artificial lipid membranes in the form of liposomes to investigate possible mechanisms for the insertion of these proteins into membranes in order to elucidate their structure and stoichiometry in the transmembrane state. We have successfully imaged the liposomes as a lipid bilayer, the S100A8/A9 protein complex in solution and the S100A8/A9 complex associating with lipid, using tapping-mode atomic force microscopy, in buffer.

The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection

PubMed Central

Haynes, Lia M.; Anderson, Larry J.

2017-01-01

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182–186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting. PMID:28671606

The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection.

PubMed

Bakre, Abhijeet A; Harcourt, Jennifer L; Haynes, Lia M; Anderson, Larry J; Tripp, Ralph A

2017-07-03

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182-186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

PubMed

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2013-01-01

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

Insight into structural requirements for selective and/or dual CXCR3 and CXCR4 allosteric modulators.

PubMed

Kolarič, Anja; Švajger, Urban; Tomašič, Tihomir; Brox, Regine; Frank, Theresa; Minovski, Nikola; Tschammer, Nuska; Anderluh, Marko

2018-05-11

Based on the previously published pyrazolopyridine-based hit compound for which negative allosteric modulation of both CXCR3 and CXCR4 receptors was disclosed, we designed, synthesized and biologically evaluated a set of novel, not only negative, but also positive allosteric modulators with preserved pyrazolopyridine core. Compound 9e is a dual negative modulator, inhibiting G protein activity of both receptors. For CXCR4 receptor para-substituted aromatic group of compounds distinguishes between negative and positive modulation. Para-methoxy substitution leads to functional antagonism, while para-chloro triggers agonism. Additionally, we discovered that chemotaxis is not completely correlated with G protein pathways. This is the first work in which we have on a series of compounds successfully demonstrated that it is possible to produce selective as well as dual-acting modulators of chemokine receptors, which is very promising for future research in the field of discovery of selective or dual modulators of chemokine receptors. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

fabp4 is central to eight obesity associated genes: a functional gene network-based polymorphic study.

PubMed

Bag, Susmita; Ramaiah, Sudha; Anbarasu, Anand

2015-01-07

Network study on genes and proteins offers functional basics of the complexity of gene and protein, and its interacting partners. The gene fatty acid-binding protein 4 (fabp4) is found to be highly expressed in adipose tissue, and is one of the most abundant proteins in mature adipocytes. Our investigations on functional modules of fabp4 provide useful information on the functional genes interacting with fabp4, their biochemical properties and their regulatory functions. The present study shows that there are eight set of candidate genes: acp1, ext2, insr, lipe, ostf1, sncg, usp15, and vim that are strongly and functionally linked up with fabp4. Gene ontological analysis of network modules of fabp4 provides an explicit idea on the functional aspect of fabp4 and its interacting nodes. The hierarchal mapping on gene ontology indicates gene specific processes and functions as well as their compartmentalization in tissues. The fabp4 along with its interacting genes are involved in lipid metabolic activity and are integrated in multi-cellular processes of tissues and organs. They also have important protein/enzyme binding activity. Our study elucidated disease-associated nsSNP prediction for fabp4 and it is interesting to note that there are four rsID׳s (rs1051231, rs3204631, rs140925685 and rs141169989) with disease allelic variation (T104P, T126P, G27D and G90V respectively). On the whole, our gene network analysis presents a clear insight about the interactions and functions associated with fabp4 gene network. Copyright © 2014 Elsevier Ltd. All rights reserved.

Markov State Models Provide Insights into Dynamic Modulation of Protein Function

PubMed Central

2015-01-01

Conspectus Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or “molecular switches” within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a theoretical toolbox for studying the effect of nonequilibrium perturbations on conformational dynamics. Considering that protein dynamics in vivo occur under nonequilibrium conditions, MSMs coupled with nonequilibrium statistical mechanics provide a way to connect cellular components to their functional environments. Nonequilibrium perturbations of protein folding MSMs reveal the presence of dynamically frozen glass-like states in their conformational landscape. These frozen states are also observed to be rich in β-sheets, which indicates their possible role in the nucleation of β-sheet rich aggregates such as those observed in amyloid-fibril formation. Finally, we describe how MSMs have been used to understand the dynamical behavior of intrinsically disordered proteins such as amyloid-β, human islet amyloid polypeptide, and p53. While certainly not a panacea for studying functional dynamics, MSMs provide a rigorous theoretical foundation for understanding complex entropically dominated processes and a convenient lens for viewing protein motions. PMID:25625937

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

Year 2 Report: Protein Function Prediction Platform

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

Zhou, C E

2012-04-27

Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fullymore » automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.« less

Allosteric Modulation of Chemoattractant Receptors

PubMed Central

Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

2016-01-01

Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

High pressure effects on protein structure and function.

PubMed

Mozhaev, V V; Heremans, K; Frank, J; Masson, P; Balny, C

1996-01-01

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

Identification of Modules in Protein-Protein Interaction Networks

NASA Astrophysics Data System (ADS)

Erten, Sinan; Koyutürk, Mehmet

In biological systems, most processes are carried out through orchestration of multiple interacting molecules. These interactions are often abstracted using network models. A key feature of cellular networks is their modularity, which contributes significantly to the robustness, as well as adaptability of biological systems. Therefore, modularization of cellular networks is likely to be useful in obtaining insights into the working principles of cellular systems, as well as building tractable models of cellular organization and dynamics. A common, high-throughput source of data on molecular interactions is in the form of physical interactions between proteins, which are organized into protein-protein interaction (PPI) networks. This chapter provides an overview on identification and analysis of functional modules in PPI networks, which has been an active area of research in the last decade.

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

PubMed Central

Suresh, S. N.; Chavalmane, Aravinda K.; DJ, Vidyadhara; Yarreiphang, Haorei; Rai, Shashank; Paul, Abhik; Clement, James P.; Alladi, Phalguni Anand; Manjithaya, Ravi

2017-01-01

ABSTRACT Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders. PMID:28350199

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans.

PubMed

Tseng, Rong-Jeng; Armstrong, Kristin R; Wang, Xiaodong; Chamberlin, Helen M

2007-11-01

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences.

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

PubMed

Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-11-27

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

Amyloid precursor protein modulates macrophage phenotype and diet-dependent weight gain

PubMed Central

Puig, Kendra L.; Brose, Stephen A.; Zhou, Xudong; Sens, Mary A.; Combs, Gerald F.; Jensen, Michael D.; Golovko, Mikhail Y.; Combs, Colin K.

2017-01-01

It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer’s disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer’s disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer’s disease but also diet-associated obesity. PMID:28262782

Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

PubMed

Zhang, Jiyong; Sadowska, Grazyna B; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Banks, William A; Stonestreet, Barbara S

2015-05-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia. © FASEB.

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

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

Asteriti, Sabrina; Dal Cortivo, Giuditta; Pontelli, Valeria

2015-06-12

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release weremore » in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.« less

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

PubMed

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

PubMed Central

Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

2015-01-01

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.

Coherent control of an opsin in living brain tissue

NASA Astrophysics Data System (ADS)

Paul, Kush; Sengupta, Parijat; Ark, Eugene D.; Tu, Haohua; Zhao, Youbo; Boppart, Stephen A.

2017-11-01

Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these proteins has been studied outside cellular environments using ultrashort tailored light pulses. However, how living cell functions can be modulated via opsins by modifying fundamental nonlinear optical properties of light interacting with the retinal chromophore has remained largely unexplored. We report the use of chirped ultrashort near-infrared pulses to modulate light-evoked ionic current from Channelrhodopsin-2 (ChR2) in brain tissue, and consequently the firing pattern of neurons, by manipulating the phase of the spectral components of the light. These results confirm that quantum coherence of the retinal-based protein system, even in a living neuron, can influence its current output, and open up the possibilities of using designer-tailored pulses for controlling molecular dynamics of opsins in living tissue to selectively enhance or suppress neuronal function for adaptive feedback-loop applications in the future.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators.

PubMed

Huryn, Donna M; Brodsky, Jeffrey L; Brummond, Kay M; Chambers, Peter G; Eyer, Benjamin; Ireland, Alex W; Kawasumi, Masaoki; Laporte, Matthew G; Lloyd, Kayla; Manteau, Baptiste; Nghiem, Paul; Quade, Bettina; Seguin, Sandlin P; Wipf, Peter

2011-04-26

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored "chemical space." Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

PubMed Central

Huryn, Donna M.; Brodsky, Jeffrey L.; Brummond, Kay M.; Chambers, Peter G.; Eyer, Benjamin; Ireland, Alex W.; Kawasumi, Masaoki; LaPorte, Matthew G.; Lloyd, Kayla; Manteau, Baptiste; Nghiem, Paul; Quade, Bettina; Seguin, Sandlin P.; Wipf, Peter

2011-01-01

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored “chemical space.” Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point. PMID:21502524

Conservation of hot regions in protein-protein interaction in evolution.

PubMed

Hu, Jing; Li, Jiarui; Chen, Nansheng; Zhang, Xiaolong

2016-11-01

The hot regions of protein-protein interactions refer to the active area which formed by those most important residues to protein combination process. With the research development on protein interactions, lots of predicted hot regions can be discovered efficiently by intelligent computing methods, while performing biology experiments to verify each every prediction is hardly to be done due to the time-cost and the complexity of the experiment. This study based on the research of hot spot residue conservations, the proposed method is used to verify authenticity of predicted hot regions that using machine learning algorithm combined with protein's biological features and sequence conservation, though multiple sequence alignment, module substitute matrix and sequence similarity to create conservation scoring algorithm, and then using threshold module to verify the conservation tendency of hot regions in evolution. This research work gives an effective method to verify predicted hot regions in protein-protein interactions, which also provides a useful way to deeply investigate the functional activities of protein hot regions. Copyright © 2016. Published by Elsevier Inc.

FOXO3a regulates BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress

PubMed Central

Kohlbrenner, Erik; Gamb, Scott I.; Guenzel, Adam J.; Klaus, Katherine; Fayyaz, Ahmed U.; Nair, K. Sreekumaran; Hajjar, Roger J.

2016-01-01

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/role-of-foxo3a-in-heart-failure/. PMID:27694219

Microarray analysis reveals key genes and pathways in Tetralogy of Fallot

PubMed Central

He, Yue-E; Qiu, Hui-Xian; Jiang, Jian-Bing; Wu, Rong-Zhou; Xiang, Ru-Lian; Zhang, Yuan-Hai

2017-01-01

The aim of the present study was to identify key genes that may be involved in the pathogenesis of Tetralogy of Fallot (TOF) using bioinformatics methods. The GSE26125 microarray dataset, which includes cardiovascular tissue samples derived from 16 children with TOF and five healthy age-matched control infants, was downloaded from the Gene Expression Omnibus database. Differential expression analysis was performed between TOF and control samples to identify differentially expressed genes (DEGs) using Student's t-test, and the R/limma package, with a log2 fold-change of >2 and a false discovery rate of <0.01 set as thresholds. The biological functions of DEGs were analyzed using the ToppGene database. The ReactomeFIViz application was used to construct functional interaction (FI) networks, and the genes in each module were subjected to pathway enrichment analysis. The iRegulon plugin was used to identify transcription factors predicted to regulate the DEGs in the FI network, and the gene-transcription factor pairs were then visualized using Cytoscape software. A total of 878 DEGs were identified, including 848 upregulated genes and 30 downregulated genes. The gene FI network contained seven function modules, which were all comprised of upregulated genes. Genes enriched in Module 1 were enriched in the following three neurological disorder-associated signaling pathways: Parkinson's disease, Alzheimer's disease and Huntington's disease. Genes in Modules 0, 3 and 5 were dominantly enriched in pathways associated with ribosomes and protein translation. The Xbox binding protein 1 transcription factor was demonstrated to be involved in the regulation of genes encoding the subunits of cytoplasmic and mitochondrial ribosomes, as well as genes involved in neurodegenerative disorders. Therefore, dysfunction of genes involved in signaling pathways associated with neurodegenerative disorders, ribosome function and protein translation may contribute to the pathogenesis of TOF. PMID:28713939

Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function

PubMed Central

Selvais, Charlotte; D'Auria, Ludovic; Tyteca, Donatienne; Perrot, Gwenn; Lemoine, Pascale; Troeberg, Linda; Dedieu, Stéphane; Noël, Agnès; Nagase, Hideaki; Henriet, Patrick; Courtoy, Pierre J.; Marbaix, Etienne; Emonard, Hervé

2011-01-01

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa α-chain) linked to a shorter transmembrane subunit (85-kDa β-chain). LRP-1 cell-surface level and function are controlled by proteolytic shedding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ∼2-fold in cholesterol. Two membrane-associated metalloproteinases were involved in LRP-1 shedding: a disintegrin and metalloproteinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1-MMP, and specific tissue inhibitor of metalloproteinases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ∼4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by cholesterol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumulation of gelatinases (MMP-2 and MMP-9) in the medium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracellular proteolytic activities.—Selvais, C., D'Auria, L., Tyteca, D., Perrot, G, Lemoine, P., Troeberg, L., Dedieu, S., Noël, A., Nagase, H., Henriet, P., Courtoy, P. J., Marbaix, E., Emonard, H. Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function. PMID:21518850

Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages

USDA-ARS?s Scientific Manuscript database

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinan...

Excavation of attractor modules for nasopharyngeal carcinoma via integrating systemic module inference with attract method.

PubMed

Jiang, T; Jiang, C-Y; Shu, J-H; Xu, Y-J

2017-07-10

The molecular mechanism of nasopharyngeal carcinoma (NPC) is poorly understood and effective therapeutic approaches are needed. This research aimed to excavate the attractor modules involved in the progression of NPC and provide further understanding of the underlying mechanism of NPC. Based on the gene expression data of NPC, two specific protein-protein interaction networks for NPC and control conditions were re-weighted using Pearson correlation coefficient. Then, a systematic tracking of candidate modules was conducted on the re-weighted networks via cliques algorithm, and a total of 19 and 38 modules were separately identified from NPC and control networks, respectively. Among them, 8 pairs of modules with similar gene composition were selected, and 2 attractor modules were identified via the attract method. Functional analysis indicated that these two attractor modules participate in one common bioprocess of cell division. Based on the strategy of integrating systemic module inference with the attract method, we successfully identified 2 attractor modules. These attractor modules might play important roles in the molecular pathogenesis of NPC via affecting the bioprocess of cell division in a conjunct way. Further research is needed to explore the correlations between cell division and NPC.

The arabidopsis polyamine transporter LHRI/AtPUT3 modulates heat responsive gene expression by regulating mRNA stability

USDA-ARS?s Scientific Manuscript database

Polyamines (PA) involve in the gene regulation by interacting with various anionic macromolecules such as DNA, RNA and proteins and modulating their structure and function. Previous studies have showed that changing in polyamine biosynthesis alters plant response to different abiotic stresses. Here,...

Modulation of low-voltage-activated T-type Ca²⁺ channels.

PubMed

Zhang, Yuan; Jiang, Xinghong; Snutch, Terrance P; Tao, Jin

2013-07-01

Low-voltage-activated T-type Ca²⁺ channels contribute to a wide variety of physiological functions, most predominantly in the nervous, cardiovascular and endocrine systems. Studies have documented the roles of T-type channels in sleep, neuropathic pain, absence epilepsy, cell proliferation and cardiovascular function. Importantly, novel aspects of the modulation of T-type channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Although there is substantial literature regarding modulation of native T-type channels, the underlying molecular mechanisms have only recently begun to be addressed. This review focuses on recent evidence that the Ca(v)3 subunits of T-type channels, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are differentially modulated by a multitude of endogenous ligands including anandamide, monocyte chemoattractant protein-1, endostatin, and redox and oxidizing agents. The review also provides an overview of recent knowledge gained concerning downstream pathways involving G-protein-coupled receptors. This article is part of a Special Issue entitled: Calcium channels. Copyright © 2012 Elsevier B.V. All rights reserved.

Oxidative stress and protein aggregation during biological aging.

PubMed

Squier, T C

2001-09-01

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Disease gene classification with metagraph representations.

PubMed

Kircali Ata, Sezin; Fang, Yuan; Wu, Min; Li, Xiao-Li; Xiao, Xiaokui

2017-12-01

Protein-protein interaction (PPI) networks play an important role in studying the functional roles of proteins, including their association with diseases. However, protein interaction networks are not sufficient without the support of additional biological knowledge for proteins such as their molecular functions and biological processes. To complement and enrich PPI networks, we propose to exploit biological properties of individual proteins. More specifically, we integrate keywords describing protein properties into the PPI network, and construct a novel PPI-Keywords (PPIK) network consisting of both proteins and keywords as two different types of nodes. As disease proteins tend to have a similar topological characteristics on the PPIK network, we further propose to represent proteins with metagraphs. Different from a traditional network motif or subgraph, a metagraph can capture a particular topological arrangement involving the interactions/associations between both proteins and keywords. Based on the novel metagraph representations for proteins, we further build classifiers for disease protein classification through supervised learning. Our experiments on three different PPI databases demonstrate that the proposed method consistently improves disease protein prediction across various classifiers, by 15.3% in AUC on average. It outperforms the baselines including the diffusion-based methods (e.g., RWR) and the module-based methods by 13.8-32.9% for overall disease protein prediction. For predicting breast cancer genes, it outperforms RWR, PRINCE and the module-based baselines by 6.6-14.2%. Finally, our predictions also turn out to have better correlations with literature findings from PubMed. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

2010-01-01

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

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response.

PubMed

Mukhopadhyay, Himadri; de Wet, Ben; Clemens, Lara; Maini, Philip K; Allard, Jun; van der Merwe, P Anton; Dushek, Omer

2016-04-26

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans

PubMed Central

van der Meer-van Kraaij, Cindy; Siezen, Roland; Kramer, Evelien; Reinders, Marjolein; Blokzijl, Hans; van der Meer, Roelof

2007-01-01

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive “top” face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents. PMID:18850182

Handling Gene and Protein Names in the Age of Bioinformatics: The Special Challenge of Secreted Multimodular Bacterial Enzymes such as the cbhA/cbh9A Gene of Clostridium thermocellum

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

Brunecky, Roman; Schwarz, Wolfgang H.; Broeker, Jannis

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with amore » respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.« less

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Computational protein design-the next generation tool to expand synthetic biology applications.

PubMed

Gainza-Cirauqui, Pablo; Correia, Bruno Emanuel

2018-05-02

One powerful approach to engineer synthetic biology pathways is the assembly of proteins sourced from one or more natural organisms. However, synthetic pathways often require custom functions or biophysical properties not displayed by natural proteins, limitations that could be overcome through modern protein engineering techniques. Structure-based computational protein design is a powerful tool to engineer new functional capabilities in proteins, and it is beginning to have a profound impact in synthetic biology. Here, we review efforts to increase the capabilities of synthetic biology using computational protein design. We focus primarily on computationally designed proteins not only validated in vitro, but also shown to modulate different activities in living cells. Efforts made to validate computational designs in cells can illustrate both the challenges and opportunities in the intersection of protein design and synthetic biology. We also highlight protein design approaches, which although not validated as conveyors of new cellular function in situ, may have rapid and innovative applications in synthetic biology. We foresee that in the near-future, computational protein design will vastly expand the functional capabilities of synthetic cells. Copyright © 2018. Published by Elsevier Ltd.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

PubMed Central

Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

2012-01-01

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane. PMID:22240901

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

PubMed

Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

2012-06-01

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C.

PubMed

Mestek, A; Hurley, J H; Bye, L S; Campbell, A D; Chen, Y; Tian, M; Liu, J; Schulman, H; Yu, L

1995-03-01

Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

Inference of domain-disease associations from domain-protein, protein-disease and disease-disease relationships.

PubMed

Zhang, Wangshu; Coba, Marcelo P; Sun, Fengzhu

2016-01-11

Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease mechanisms.

Claudins and the Modulation of Tight Junction Permeability

PubMed Central

Günzel, Dorothee

2013-01-01

Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions. PMID:23589827

Integrative Analysis of GWASs, Human Protein Interaction, and Gene Expression Identified Gene Modules Associated With BMDs

PubMed Central

He, Hao; Zhang, Lei; Li, Jian; Wang, Yu-Ping; Zhang, Ji-Gang; Shen, Jie; Guo, Yan-Fang

2014-01-01

Context: To date, few systems genetics studies in the bone field have been performed. We designed our study from a systems-level perspective by integrating genome-wide association studies (GWASs), human protein-protein interaction (PPI) network, and gene expression to identify gene modules contributing to osteoporosis risk. Methods: First we searched for modules significantly enriched with bone mineral density (BMD)-associated genes in human PPI network by using 2 large meta-analysis GWAS datasets through a dense module search algorithm. One included 7 individual GWAS samples (Meta7). The other was from the Genetic Factors for Osteoporosis Consortium (GEFOS2). One was assigned as a discovery dataset and the other as an evaluation dataset, and vice versa. Results: In total, 42 modules and 129 modules were identified significantly in both Meta7 and GEFOS2 datasets for femoral neck and spine BMD, respectively. There were 3340 modules identified for hip BMD only in Meta7. As candidate modules, they were assessed for the biological relevance to BMD by gene set enrichment analysis in 2 expression profiles generated from circulating monocytes in subjects with low versus high BMD values. Interestingly, there were 2 modules significantly enriched in monocytes from the low BMD group in both gene expression datasets (nominal P value <.05). Two modules had 16 nonredundant genes. Functional enrichment analysis revealed that both modules were enriched for genes involved in Wnt receptor signaling and osteoblast differentiation. Conclusion: We highlighted 2 modules and novel genes playing important roles in the regulation of bone mass, providing important clues for therapeutic approaches for osteoporosis. PMID:25119315

Investigating neuronal function with optically controllable proteins

PubMed Central

Zhou, Xin X.; Pan, Michael; Lin, Michael Z.

2015-01-01

In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain. PMID:26257603

CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

PubMed Central

Garrett, Roger A.; Shah, Shiraz A.; Erdmann, Susanne; Liu, Guannan; Mousaei, Marzieh; León-Sobrino, Carlos; Peng, Wenfang; Gudbergsdottir, Soley; Deng, Ling; Vestergaard, Gisle; Peng, Xu; She, Qunxin

2015-01-01

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. PMID:25764276

Lactoferrin modulation of BCG-infected dendritic cell functions

PubMed Central

Hwang, Shen-An

2009-01-01

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-2] and increased regulatory cytokine, transforming growth factor-β1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-γ activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3+CD4+ splenocytes, as defined by increasing IFN-γ production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-γ producing CD3+CD4+CD62L− splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells. PMID:19692539

The yeast telomerase recruitment module requires a specific RNA architecture.

PubMed

Laterreur, Nancy; Lemieux, Bruno; Neumann, Hannah; Berger-Dancause, Jean-Christophe; Lafontaine, Daniel; Wellinger, Raymund J

2018-05-18

Telomerases are ribonucleoprotein (RNP) enzymes that are related to reverse transcriptases. While they maintain genome stability, their composition varies significantly between species. Yeast telomerase RNPs contain an RNA that is comparatively large and its overall folding shows long helical segments with distal functional parts. Here we investigated the essential stem IVc module of the budding yeast telomerase RNA, called Tlc1. The distal part of stem IVc includes a conserved sequence element CS2a and structurally conserved features to which bind the Pop1/Pop6/Pop7 proteins and which together function analogously to the P3 domains of the RNase P/MRP RNPs. A more proximal bulged stem with the CS2 element is thought to associate with Est1. Previous data showed that changes in CS2a cause a loss of all of the proteins, not just the Pop-proteins, from stem IVc. The results here show that the association of Est1 with stem IVc indeed requires both the proximal bulged stem and the presence of the Tlc1 P3 domain with the associated Pop-proteins. Separating the P3-domain from the Est1 binding site by inserting only 2 base pairs into the helical stem between the two sites causes a complete loss of Est1 from the RNP and hence a telomerase-negative phenotype in vivo. Still, the distal P3 domain with the associated Pop-proteins remains intact. Moreover, the P3 domain also ensures Est2 stability on the RNP independently of the Est1 association. Therefore, the recruitment module of the Tlc1 RNA requires a very tight architectural organization for telomerase function in vivo. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Altered Protein Interactions of the Endogenous Interactome of PTPIP51 towards MAPK Signaling

PubMed Central

Brobeil, Alexander; Chehab, Rajaa; Dietel, Eric; Gattenlöhner, Stefan; Wimmer, Monika

2017-01-01

Protein–protein interactions play a pivotal role in normal cellular functions as well as in carcinogenesis. The protein–protein interactions form functional clusters during signal transduction. To elucidate the fine calibration of the protein–protein interactions of protein tyrosine phosphatase interacting protein 51 (PTPIP51) a small molecule drug, namely LDC-3, directly targeting PTPIP51 is now available. Therefore, LDC-3 allows for the studying of the regulation of the endogenous interactome by modulating PTPIP51 binding capacity. Small interfering ribonucleic acid (siRNA) experiments show that the modification in PTPIP51 binding capacity is induced by LDC-3. Application of LDC-3 annuls the known regulatory phosphorylation mechanisms for PTPIP51 and consequently, significantly alters the assembly of the PTPIP51 associated protein complexes. The treatment of human keratinocytes (HaCaT cells) with LDC-3 induces an altered protein–protein interaction profile of the endogenous interactome of PTPIP51. In addition, LDC-3 stabilizes PTPIP51 within a mitogen activated protein kinase (MAPK) complex composed of Raf-1 and the scaffold protein 14-3-3, independent of the phosphorylation status of PTPIP51. Of note, under LDC-3 treatment the regulatory function of the PTP1B on PTPIP51 fails to impact the PTPIP51 interaction characteristics, as reported for the HaCaT cell line. In summary, LDC-3 gives the unique opportunity to directly modulate PTPIP51 in malignant cells, thus targeting potential dysregulated signal transduction pathways such as the MAPK cascade. The provided data give critical insights in the therapeutic potential of PTPIP51 protein interactions and thus are basic for possible targeted therapy regimens. PMID:28754031

Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

DOEpatents

Oda, Michael N [Benicia, CA; Forte, Trudy M [Berkeley, CA

2007-05-29

Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

Supramolecularly Engineered Circular Bivalent Aptamer for Enhanced Functional Protein Delivery.

PubMed

Jiang, Ying; Pan, Xiaoshu; Chang, Jin; Niu, Weijia; Hou, Weijia; Kuai, Hailan; Zhao, Zilong; Liu, Ji; Wang, Ming; Tan, Weihong

2018-06-06

Circular bivalent aptamers (cb-apt) comprise an emerging class of chemically engineered aptamers with substantially improved stability and molecular recognition ability. Its therapeutic application, however, is challenged by the lack of functional modules to control the interactions of cb-apt with therapeutics. We present the design of a β-cyclodextrin-modified cb-apt (cb-apt-βCD) and its supramolecular interaction with molecular therapeutics via host-guest chemistry for targeted intracellular delivery. The supramolecular ensemble exhibits high serum stability and enhanced intracellular delivery efficiency compared to a monomeric aptamer. The cb-apt-βCD ensemble delivers green fluorescent protein into targeted cells with efficiency as high as 80%, or cytotoxic saporin to efficiently inhibit tumor cell growth. The strategy of conjugating βCD to cb-apt, and subsequently modulating the supramolecular chemistry of cb-apt-βCD, provides a general platform to expand and diversify the function of aptamers, enabling new biological and therapeutic applications.

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

PubMed Central

Siebert, Adam P.; Ma, Zhongming; Grevet, Jeremy D.; Demuro, Angelo; Parker, Ian; Foskett, J. Kevin

2013-01-01

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca2+ concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties. PMID:23300080

The muscle protein synthetic response to food ingestion.

PubMed

Gorissen, Stefan H M; Rémond, Didier; van Loon, Luc J C

2015-11-01

Preservation of skeletal muscle mass is of great importance for maintaining both metabolic health and functional capacity. Muscle mass maintenance is regulated by the balance between muscle protein breakdown and synthesis rates. Both muscle protein breakdown and synthesis rates have been shown to be highly responsive to physical activity and food intake. Food intake, and protein ingestion in particular, directly stimulates muscle protein synthesis rates. The postprandial muscle protein synthetic response to feeding is regulated on a number of levels, including dietary protein digestion and amino acid absorption, splanchnic amino acid retention, postprandial insulin release, skeletal muscle tissue perfusion, amino acid uptake by muscle, and intramyocellular signaling. The postprandial muscle protein synthetic response to feeding is blunted in many conditions characterized by skeletal muscle loss, such as aging and muscle disuse. Therefore, it is important to define food characteristics that modulate postprandial muscle protein synthesis. Previous work has shown that the muscle protein synthetic response to feeding can be modulated by changing the amount of protein ingested, the source of dietary protein, as well as the timing of protein consumption. Most of this work has studied the postprandial response to the ingestion of isolated protein sources. Only few studies have investigated the postprandial muscle protein synthetic response to the ingestion of protein dense foods, such as dairy and meat. The current review will focus on the capacity of proteins and protein dense food products to stimulate postprandial muscle protein synthesis and identifies food characteristics that may modulate the anabolic properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

PubMed

Miller, Justin M; Enemark, Eric J

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

PubMed Central

Campiglio, Marta; Flucher, Bernhard E

2015-01-01

Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

Ror receptor tyrosine kinases: orphans no more.

PubMed

Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

2008-11-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

The Prion Protein Modulates A-type K+ Currents Mediated by Kv4.2 Complexes through Dipeptidyl Aminopeptidase-like Protein 6*

PubMed Central

Mercer, Robert C. C.; Ma, Li; Watts, Joel C.; Strome, Robert; Wohlgemuth, Serene; Yang, Jing; Cashman, Neil R.; Coulthart, Michael B.; Schmitt-Ulms, Gerold; Jhamandas, Jack H.; Westaway, David

2013-01-01

Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K+ channels, we assessed the impact of WT and mutant PrPC upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrPC, in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrPC was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability. PMID:24225951

Active transport of vesicles in neurons is modulated by mechanical tension.

PubMed

Ahmed, Wylie W; Saif, Taher A

2014-03-27

Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics.

Active transport of vesicles in neurons is modulated by mechanical tension

PubMed Central

Ahmed, Wylie W.; Saif, Taher A.

2014-01-01

Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics. PMID:24670781

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

PubMed

Kitamura, Koichiro; Komatsu, Masayuki; Biyani, Madhu; Futakami, Masae; Kawakubo, Tomoyo; Yamamoto, Kenji; Nishigaki, Koichi

2012-12-01

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein.

PubMed

Vander Wood, Drew A; Keatinge-Clay, Adrian T

2018-06-01

Here, the term "module" is redefined for trans-acyltransferase (trans-AT) assembly lines to agree with how its domains cooperate and evolutionarily co-migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans-AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co-evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not-in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering. © 2018 Wiley Periodicals, Inc.

Evolutionary divergence of chloroplast FAD synthetase proteins

PubMed Central

2010-01-01

Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity. PMID:20955574

Advances in molecular engineering of carbohydrate-binding modules.

PubMed

Armenta, Silvia; Moreno-Mendieta, Silvia; Sánchez-Cuapio, Zaira; Sánchez, Sergio; Rodríguez-Sanoja, Romina

2017-09-01

Carbohydrate-binding modules (CBMs) are non-catalytic domains that are generally appended to carbohydrate-active enzymes. CBMs have a broadly conserved structure that allows recognition of a notable variety of carbohydrates, in both their soluble and insoluble forms, as well as in their alpha and beta conformations and with different types of bonds or substitutions. This versatility suggests a high functional plasticity that is not yet clearly understood, in spite of the important number of studies relating protein structure and function. Several studies have explored the flexibility of these systems by changing or improving their specificity toward substrates of interest. In this review, we examine the molecular strategies used to identify CBMs with novel or improved characteristics. The impact of the spatial arrangement of the functional amino acids of CBMs is discussed in terms of unexpected new functions that are not related to the original biological roles of the enzymes. Proteins 2017; 85:1602-1617. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

PubMed Central

Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

2015-01-01

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

Phosducin-like protein: an ethanol-responsive potential modulator of guanine nucleotide-binding protein function.

PubMed

Miles, M F; Barhite, S; Sganga, M; Elliott, M

1993-11-15

Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein beta gamma subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern hybridization. We have identified four forms of PhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus. Treatment of NG108-15 cells for 24 hr with concentrations of ethanol seen in actively drinking alcoholics (25-100 mM) causes up to a 3-fold increase in PhLP mRNA levels. Induction of PhLP by ethanol could account for at least some of the widespread alterations in signal transduction and G-protein function that are known to occur with chronic exposure to ethanol.

Discovering perturbation of modular structure in HIV progression by integrating multiple data sources through non-negative matrix factorization.

PubMed

Ray, Sumanta; Maulik, Ujjwal

2016-12-20

Detecting perturbation in modular structure during HIV-1 disease progression is an important step to understand stage specific infection pattern of HIV-1 virus in human cell. In this article, we proposed a novel methodology on integration of multiple biological information to identify such disruption in human gene module during different stages of HIV-1 infection. We integrate three different biological information: gene expression information, protein-protein interaction information and gene ontology information in single gene meta-module, through non negative matrix factorization (NMF). As the identified metamodules inherit those information so, detecting perturbation of these, reflects the changes in expression pattern, in PPI structure and in functional similarity of genes during the infection progression. To integrate modules of different data sources into strong meta-modules, NMF based clustering is utilized here. Perturbation in meta-modular structure is identified by investigating the topological and intramodular properties and putting rank to those meta-modules using a rank aggregation algorithm. We have also analyzed the preservation structure of significant GO terms in which the human proteins of the meta-modules participate. Moreover, we have performed an analysis to show the change of coregulation pattern of identified transcription factors (TFs) over the HIV progression stages.

PTEN: Multiple Functions in Human Malignant Tumors.

PubMed

Milella, Michele; Falcone, Italia; Conciatori, Fabiana; Cesta Incani, Ursula; Del Curatolo, Anais; Inzerilli, Nicola; Nuzzo, Carmen M A; Vaccaro, Vanja; Vari, Sabrina; Cognetti, Francesco; Ciuffreda, Ludovica

2015-01-01

PTEN is the most important negative regulator of the PI3K signaling pathway. In addition to its canonical, PI3K inhibition-dependent functions, PTEN can also function as a tumor suppressor in a PI3K-independent manner. Indeed, the PTEN network regulates a broad spectrum of biological functions, modulating the flow of information from membrane-bound growth factor receptors to nuclear transcription factors, occurring in concert with other tumor suppressors and oncogenic signaling pathways. PTEN acts through its lipid and protein phosphatase activity and other non-enzymatic mechanisms. Studies conducted over the past 10 years have expanded our understanding of the biological role of PTEN, showing that in addition to its ability to regulate proliferation and cell survival, it also plays an intriguing role in regulating genomic stability, cell migration, stem cell self-renewal, and tumor microenvironment. Changes in PTEN protein levels, location, and enzymatic activity through various molecular mechanisms can generate a continuum of functional PTEN levels in inherited syndromes, sporadic cancers, and other diseases. PTEN activity can indeed, be modulated by mutations, epigenetic silencing, transcriptional repression, aberrant protein localization, and post-translational modifications. This review will discuss our current understanding of the biological role of PTEN, how PTEN expression and activity are regulated, and the consequences of PTEN dysregulation in human malignant tumors.

PTEN: Multiple Functions in Human Malignant Tumors

PubMed Central

Milella, Michele; Falcone, Italia; Conciatori, Fabiana; Cesta Incani, Ursula; Del Curatolo, Anais; Inzerilli, Nicola; Nuzzo, Carmen M. A.; Vaccaro, Vanja; Vari, Sabrina; Cognetti, Francesco; Ciuffreda, Ludovica

2015-01-01

PTEN is the most important negative regulator of the PI3K signaling pathway. In addition to its canonical, PI3K inhibition-dependent functions, PTEN can also function as a tumor suppressor in a PI3K-independent manner. Indeed, the PTEN network regulates a broad spectrum of biological functions, modulating the flow of information from membrane-bound growth factor receptors to nuclear transcription factors, occurring in concert with other tumor suppressors and oncogenic signaling pathways. PTEN acts through its lipid and protein phosphatase activity and other non-enzymatic mechanisms. Studies conducted over the past 10 years have expanded our understanding of the biological role of PTEN, showing that in addition to its ability to regulate proliferation and cell survival, it also plays an intriguing role in regulating genomic stability, cell migration, stem cell self-renewal, and tumor microenvironment. Changes in PTEN protein levels, location, and enzymatic activity through various molecular mechanisms can generate a continuum of functional PTEN levels in inherited syndromes, sporadic cancers, and other diseases. PTEN activity can indeed, be modulated by mutations, epigenetic silencing, transcriptional repression, aberrant protein localization, and post-translational modifications. This review will discuss our current understanding of the biological role of PTEN, how PTEN expression and activity are regulated, and the consequences of PTEN dysregulation in human malignant tumors. PMID:25763354

A theoretical model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: From biostructural pathology to specie

NASA Astrophysics Data System (ADS)

Villoutreix, Bruno O.; Teleman, Olle; Dahlbäck, Björn

1997-05-01

Protein S (PS), which functions as a species-specific anticoagulant cofactor to activated protein C (APC), is a mosaic protein that interacts with the phospholipid membrane via its γ-carboxyglutamate-rich (Gla) module. This module is followed by the thrombin-sensitive region (TSR), sensitive to thrombin cleavage, four epidermal growth factor (EGF)-like modules and a last region referred to as the sex hormone binding globulin (SHBG) domain. Of these, the TSR and the first EGF-like regions have been shown to be important for the species-specific interaction with APC. Difficulties in crystallising PS have so far hindered its study at the atomic level. Here, we report theoretical models for the Gla and EGF-1 modules of human PS constructed using prothrombin and factor X experimental structures. The TSR was built interactively. Analysis of the model linked with the large body of biochemical literature on PS and related proteins leads to suggestions that (i) the TSR stabilises the calcium-loaded Gla module through hydrophobic and ionic interactions and its conformation depends on the presence of the Gla module; (ii) the TSR does not form a calcium binding site but is protected from thrombin cleavage in the calcium-loaded form owing to short secondary structure elements and close contact with the Gla module; (iii) the PS missense mutations in this region are consistent with the structural data, except in one case which needs further investigation; and (iv) the two PS `faces' involving regions of residues Arg49-Gln52-Lys97 (TSR-EGF-1) and Thr103-Pro106 (EGF-1) may be involved in species-specific interactions with APC as they are richer in nonconservative substitution when comparing human and bovine protein S. This preliminary model helps to plan future experiments and the resulting data will be used to further validate and optimise the present structure.

A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

PubMed

Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

2009-01-02

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

Probiotic Modulation of Innate Cell Pathogen Sensing and Signaling Events

PubMed Central

Llewellyn, Amy; Foey, Andrew

2017-01-01

There is a growing body of evidence documenting probiotic bacteria to have a beneficial effect to the host through their ability to modulate the mucosal immune system. Many probiotic bacteria can be considered to act as either immune activators or immune suppressors, which have appreciable influence on homeostasis, inflammatory- and suppressive-immunopathology. What is becoming apparent is the ability of these probiotics to modulate innate immune responses via direct or indirect effects on the signaling pathways that drive these activatory or suppressive/tolerogenic mechanisms. This review will focus on the immunomodulatory role of probiotics on signaling pathways in innate immune cells: from positive to negative regulation associated with innate immune cells driving gut mucosal functionality. Research investigations have shown probiotics to modulate innate functionality in many ways including, receptor antagonism, receptor expression, binding to and expression of adaptor proteins, expression of negative regulatory signal molecules, induction of micro-RNAs, endotoxin tolerisation and finally, the secretion of immunomodulatory proteins, lipids and metabolites. The detailed understanding of the immunomodulatory signaling effects of probiotic strains will facilitate strain-specific selective manipulation of innate cell signal mechanisms in the modulation of mucosal adjuvanticity, immune deviation and tolerisation in both healthy subjects and patients with inflammatory and suppressive pathology. PMID:29065562

On the detection of functionally coherent groups of protein domains with an extension to protein annotation

PubMed Central

McLaughlin, William A; Chen, Ken; Hou, Tingjun; Wang, Wei

2007-01-01

Background Protein domains coordinate to perform multifaceted cellular functions, and domain combinations serve as the functional building blocks of the cell. The available methods to identify functional domain combinations are limited in their scope, e.g. to the identification of combinations falling within individual proteins or within specific regions in a translated genome. Further effort is needed to identify groups of domains that span across two or more proteins and are linked by a cooperative function. Such functional domain combinations can be useful for protein annotation. Results Using a new computational method, we have identified 114 groups of domains, referred to as domain assembly units (DASSEM units), in the proteome of budding yeast Saccharomyces cerevisiae. The units participate in many important cellular processes such as transcription regulation, translation initiation, and mRNA splicing. Within the units the domains were found to function in a cooperative manner; and each domain contributed to a different aspect of the unit's overall function. The member domains of DASSEM units were found to be significantly enriched among proteins contained in transcription modules, defined as genes sharing similar expression profiles and presumably similar functions. The observation further confirmed the functional coherence of DASSEM units. The functional linkages of units were found in both functionally characterized and uncharacterized proteins, which enabled the assessment of protein function based on domain composition. Conclusion A new computational method was developed to identify groups of domains that are linked by a common function in the proteome of Saccharomyces cerevisiae. These groups can either lie within individual proteins or span across different proteins. We propose that the functional linkages among the domains within the DASSEM units can be used as a non-homology based tool to annotate uncharacterized proteins. PMID:17937820

Differential network analysis reveals the genome-wide landscape of estrogen receptor modulation in hormonal cancers

PubMed Central

Hsiao, Tzu-Hung; Chiu, Yu-Chiao; Hsu, Pei-Yin; Lu, Tzu-Pin; Lai, Liang-Chuan; Tsai, Mong-Hsun; Huang, Tim H.-M.; Chuang, Eric Y.; Chen, Yidong

2016-01-01

Several mutual information (MI)-based algorithms have been developed to identify dynamic gene-gene and function-function interactions governed by key modulators (genes, proteins, etc.). Due to intensive computation, however, these methods rely heavily on prior knowledge and are limited in genome-wide analysis. We present the modulated gene/gene set interaction (MAGIC) analysis to systematically identify genome-wide modulation of interaction networks. Based on a novel statistical test employing conjugate Fisher transformations of correlation coefficients, MAGIC features fast computation and adaption to variations of clinical cohorts. In simulated datasets MAGIC achieved greatly improved computation efficiency and overall superior performance than the MI-based method. We applied MAGIC to construct the estrogen receptor (ER) modulated gene and gene set (representing biological function) interaction networks in breast cancer. Several novel interaction hubs and functional interactions were discovered. ER+ dependent interaction between TGFβ and NFκB was further shown to be associated with patient survival. The findings were verified in independent datasets. Using MAGIC, we also assessed the essential roles of ER modulation in another hormonal cancer, ovarian cancer. Overall, MAGIC is a systematic framework for comprehensively identifying and constructing the modulated interaction networks in a whole-genome landscape. MATLAB implementation of MAGIC is available for academic uses at https://github.com/chiuyc/MAGIC. PMID:26972162

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

PubMed Central

Ainsztein, Alexandra M.; Kandels-Lewis, Stefanie E.; Mackay, Alastair M.; Earnshaw, William C.

1998-01-01

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle. PMID:9864353

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

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

Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana

2008-02-20

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defectivemore » clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.« less

Protein-protein recognition control by modulating electrostatic interactions.

PubMed

Han, Song; Yin, Shijin; Yi, Hong; Mouhat, Stéphanie; Qiu, Su; Cao, Zhijian; Sabatier, Jean-Marc; Wu, Yingliang; Li, Wenxin

2010-06-04

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

PubMed

Rauschenberger, Lisa; Staar, Doreen; Thom, Kathleen; Scharf, Christian; Venz, Simone; Homuth, Georg; Schlüter, Rabea; Brandenburg, Lars-Ove; Ziegler, Patrick; Zimmermann, Uwe; Weitschies, Werner; Völker, Uwe; Lendeckel, Uwe; Walther, Reinhard; Burchardt, Martin; Stope, Matthias B

2016-03-01

Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth. © 2015 Wiley Periodicals, Inc.

FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis

PubMed Central

Wang, Pei-Jen; Yang, Wen-Kai; Lin, Chia-Hao; Hwang, Hau-Hsuan; Lee, Tsung-Han

2017-01-01

FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates. PMID:28848450

Hierarchical cortical transcriptome disorganization in autism.

PubMed

Lombardo, Michael V; Courchesne, Eric; Lewis, Nathan E; Pramparo, Tiziano

2017-01-01

Autism spectrum disorders (ASD) are etiologically heterogeneous and complex. Functional genomics work has begun to identify a diverse array of dysregulated transcriptomic programs (e.g., synaptic, immune, cell cycle, DNA damage, WNT signaling, cortical patterning and differentiation) potentially involved in ASD brain abnormalities during childhood and adulthood. However, it remains unclear whether such diverse dysregulated pathways are independent of each other or instead reflect coordinated hierarchical systems-level pathology. Two ASD cortical transcriptome datasets were re-analyzed using consensus weighted gene co-expression network analysis (WGCNA) to identify common co-expression modules across datasets. Linear mixed-effect models and Bayesian replication statistics were used to identify replicable differentially expressed modules. Eigengene network analysis was then utilized to identify between-group differences in how co-expression modules interact and cluster into hierarchical meta-modular organization. Protein-protein interaction analyses were also used to determine whether dysregulated co-expression modules show enhanced interactions. We find replicable evidence for 10 gene co-expression modules that are differentially expressed in ASD cortex. Rather than being independent non-interacting sources of pathology, these dysregulated co-expression modules work in synergy and physically interact at the protein level. These systems-level transcriptional signals are characterized by downregulation of synaptic processes coordinated with upregulation of immune/inflammation, response to other organism, catabolism, viral processes, translation, protein targeting and localization, cell proliferation, and vasculature development. Hierarchical organization of meta-modules (clusters of highly correlated modules) is also highly affected in ASD. These findings highlight that dysregulation of the ASD cortical transcriptome is characterized by the dysregulation of multiple coordinated transcriptional programs producing synergistic systems-level effects that cannot be fully appreciated by studying the individual component biological processes in isolation.

PASTA in Penicillin Binding Proteins and Serine/Threonine Kinases: A Recipe of Structural, Dynamic and Binding Properties.

PubMed

Calvanese, Luisa; Falcigno, Lucia; Squeglia, Flavia; D'Auria, Gabriella; Berisio, Rita

2017-11-24

Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Repulsive Guidance Molecules (RGMs) and Neogenin in Bone Morphogenetic Protein (BMP) signaling

PubMed Central

Tian, Chenxi; Liu, Jun

2015-01-01

Summary Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGFβ) superfamily. BMPs mediate a highly conserved signal transduction cascade through the type I and type II serine/threonine kinase receptors and intracellular Smad proteins. The BMP pathway regulates multiple developmental and homeostatic processes. Mutations in this pathway can cause various diseases in humans, such as skeletal disorders, cardiovascular diseases and various cancers. Multiple levels of regulation, including extracellular regulation, help to ensure proper spatiotemporal control of BMP signaling in the right cellular context. The family of repulsive guidance molecules (RGMs) and the type I trans-membrane protein neogenin, a paralog of DCC (Deleted in Colorectal Cancer), have been implicated in modulating the BMP pathway. In this review, we discuss the properties and functions of RGM proteins and neogenin, focusing on their roles in the modulation of BMP signal transduction. PMID:23740870

Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

PubMed

Moretti, Marino; Wang, Lei; Grognet, Pierre; Lanver, Daniel; Link, Hannes; Kahmann, Regine

2017-09-01

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions. © 2017 John Wiley & Sons Ltd.

Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

PubMed

Sivagurunathan, Suganya; Palanisamy, Karthikka; Arunachalam, Jayamuruga Pandian; Chidambaram, Subbulakshmi

2017-03-01

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight*

PubMed Central

Byerly, Mardi S.; Petersen, Pia S.; Ramamurthy, Santosh; Seldin, Marcus M.; Lei, Xia; Provost, Elayne; Wei, Zhikui; Ronnett, Gabriele V.; Wong, G. William

2014-01-01

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance. PMID:24366864

Selective androgen receptor modulators as function promoting therapies.

PubMed

Bhasin, Shalender; Jasuja, Ravi

2009-05-01

The past decade has witnessed an unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Although steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5alpha-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with androgen receptor (AR) contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand-binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis.

Selective Androgen Receptor Modulators (SARMs) as Function Promoting Therapies

PubMed Central

Bhasin, Shalender; Jasuja, Ravi

2010-01-01

Purpose of review The last decade has witnessed unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Recent Findings While steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5α-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with AR contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. Summary SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis. PMID:19357508

Aquaporins: important but elusive drug targets

PubMed Central

Verkman, Alan S.; Anderson, Marc O.; Papadopoulos, Marios C.

2014-01-01

The aquaporins (AQPs) are a family of small, integral membrane proteins that facilitate water transport across the plasma membranes of cells in response to osmotic gradients. Data from knockout mice support the involvement of AQPs in epithelial fluid secretion, cell migration, brain oedema and adipocyte metabolism, which suggests that modulation of AQP function or expression could have therapeutic potential in oedema, cancer, obesity, brain injury, glaucoma and several other conditions. Moreover, loss-of-function mutations in human AQPs cause congenital cataracts (AQP0) and nephrogenic diabetes insipidus (AQP2), and autoantibodies against AQP4 cause the autoimmune demyelinating disease neuromyelitis optica. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators include the druggability of the target and the suitability of the assay methods used to identify modulators. PMID:24625825

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

PubMed

Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

2015-11-01

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program.

PubMed

Tasseff, Ryan; Jensen, Holly A; Congleton, Johanna; Dai, David; Rogers, Katharine V; Sagar, Adithya; Bunaciu, Rodica P; Yen, Andrew; Varner, Jeffrey D

2017-10-30

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

The DEAD-box helicase eIF4A: paradigm or the odd one out?

PubMed

Andreou, Alexandra Z; Klostermeier, Dagmar

2013-01-01

DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein.

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

PubMed

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

2018-04-01

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

Engineering Proteins for Thermostability with iRDP Web Server

PubMed Central

Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C. G.

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements. PMID:26436543

Engineering Proteins for Thermostability with iRDP Web Server.

PubMed

Panigrahi, Priyabrata; Sule, Manas; Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C G

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

Molecular imaging of drug-modulated protein-protein interactions in living subjects.

PubMed

Paulmurugan, Ramasamy; Massoud, Tarik F; Huang, Jing; Gambhir, Sanjiv S

2004-03-15

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

Molecular Mechanics and Dynamics Characterization of an "in silico" Mutated Protein: A Stand-Alone Lab Module or Support Activity for "in vivo" and "in vitro" Analyses of Targeted Proteins

ERIC Educational Resources Information Center

Chiang, Harry; Robinson, Lucy C.; Brame, Cynthia J.; Messina, Troy C.

2013-01-01

Over the past 20 years, the biological sciences have increasingly incorporated chemistry, physics, computer science, and mathematics to aid in the development and use of mathematical models. Such combined approaches have been used to address problems from protein structure-function relationships to the workings of complex biological systems.…

In Women with Previous Pregnancy Hypertension, Levels of Cardiovascular Risk Biomarkers May Be Modulated by Haptoglobin Polymorphism

PubMed Central

Clara Bicho, Maria; Areias, Maria José; Rebelo, Irene

2014-01-01

Preeclampsia (PE) may affect the risk for future cardiovascular disease. Haptoglobin (Hp), an acute phase protein with functional genetic polymorphism, synthesized in the hepatocyte and in many peripheral tissues secondary of oxidative stress of PE, may modulate that risk through the antioxidant, angiogenic, and anti-inflammatory differential effects of their genotypes. We performed a prospective study in 352 women aged 35 ± 5.48 years, which 165 had previous PE, 2 to 16 years ago. We studied demographic, anthropometric, and haemodynamic biomarkers such as C-reactive protein (CRP), myeloperoxidase (MPO), and nitric oxide metabolites (total and nitrites), and others associated with liver function (AST and ALT) and lipid profile (total LDL and cholesterol HDL, non-HDL, and apolipoproteins A and B). Finally, we study the influence of Hp genetic polymorphism on all these biomarkers and as a predisposing factor for PE and its remote cardiovascular disease prognosis. Previously preeclamptic women either hypertensive or normotensive presented significant differences in those risk biomarkers (MPO, nitrites, and ALT), whose variation may be modulated by Hp 1/2 functional genetic polymorphism. The history of PE may be relevant, in association with these biomarkers to the cardiovascular risk in premenopausal women. PMID:25101128

Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

PubMed

Bruntz, Ronald C; Lindsley, Craig W; Brown, H Alex

2014-10-01

Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Proteomic profiles in hyperandrogenic syndromes.

PubMed

Misiti, S; Stigliano, A; Borro, M; Gentile, G; Michienzi, S; Cerquetti, L; Bucci, B; Argese, N; Brunetti, E; Simmaco, M; Toscano, V

2010-03-01

Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module

PubMed Central

Selkrig, Joel; Belousoff, Matthew J.; Headey, Stephen J.; Heinz, Eva; Shiota, Takuya; Shen, Hsin-Hui; Beckham, Simone A.; Bamert, Rebecca S.; Phan, Minh-Duy; Schembri, Mark A.; Wilce, Matthew C.J.; Scanlon, Martin J.; Strugnell, Richard A.; Lithgow, Trevor

2015-01-01

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes. PMID:26243377

The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: Implications for the development of HBc-targeting antivirals.

PubMed

Diab, Ahmed; Foca, Adrien; Zoulim, Fabien; Durantel, David; Andrisani, Ourania

2018-01-01

Virally encoded proteins have evolved to perform multiple functions, and the core protein (HBc) of the hepatitis B virus (HBV) is a perfect example. While HBc is the structural component of the viral nucleocapsid, additional novel functions for the nucleus-localized HBc have recently been described. These results extend for HBc, beyond its structural role, a regulatory function in the viral life cycle and potentially a role in pathogenesis. In this article, we review the diverse roles of HBc in HBV replication and pathogenesis, emphasizing how the unique structure of this protein is key to its various functions. We focus in particular on recent advances in understanding the significance of HBc phosphorylations, its interaction with host proteins and the role of HBc in regulating the transcription of host genes. We also briefly allude to the emerging niche for new direct-acting antivirals targeting HBc, known as Core (protein) Allosteric Modulators (CAMs). Copyright © 2017 Elsevier B.V. All rights reserved.

Protein Kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity

PubMed Central

Sundram, Vasudha; Ganju, Aditya; Hughes, Joshua E.; Khan, Sheema; Chauhan, Subhash C.; Jaggi, Meena

2014-01-01

Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells. PMID:25149539

Structure and Biophysics of CBFβ/RUNX and Its Translocation Products.

PubMed

Tahirov, Tahir H; Bushweller, John

2017-01-01

The core binding factor (CBF) transcription factor is somewhat unique in that it is composed of a DNA binding RUNX subunit (RUNX1, 2, or 3) and a non-DNA binding CBFβ subunit, which modulates RUNX protein activity by modulating the auto-inhibition of the RUNX subunits. Since the discovery of this fascinating transcription factor more than 20 years ago, there has been a robust effort to characterize the structure as well as the biochemical properties of CBF. More recently, these efforts have also extended to the fusion proteins that arise from the subunits of CBF in leukemia. This chapter highlights the work of numerous labs which has provided a detailed understanding of the structure and function of this transcription factor and its fusion proteins.

Sirtuins, Cell Senescence, and Vascular Aging.

PubMed

Kida, Yujiro; Goligorsky, Michael S

2016-05-01

The sirtuins (SIRTs) constitute a class of proteins with nicotinamide adenine dinucleotide-dependent deacetylase or adenosine diphosphate-ribosyltransferase activity. Seven SIRT family members have been identified in mammals, from SIRT1, the best studied for its role in vascular aging, to SIRT7. SIRT1 and SIRT2 are localized in the nucleus and cytoplasm. SIRT3, SIRT4, and SIRT5 are mitochondrial, and SIRT6 and SIRT7 are nuclear. Extensive studies have clearly revealed that SIRT proteins regulate diverse cell functions and responses to stressors. Vascular aging involves the aging process (senescence) of endothelial and vascular smooth muscle cells. Two types of cell senescence have been identified: (1) replicative senescence with telomere attrition; and (2) stress-induced premature senescence without telomere involvement. Both types of senescence induce vascular cell growth arrest and loss of vascular homeostasis, and contribute to the initiation and progression of cardiovascular diseases. Previous mechanistic studies have revealed in detail that SIRT1, SIRT3, and SIRT6 show protective functions against vascular aging, and definite vascular function of other SIRTs is under investigation. Thus, direct SIRT modulation and nicotinamide adenine dinucleotide stimulation of SIRT are promising candidates for cardiovascular disease therapy. A small number of pilot studies have been conducted to assess SIRT modulation in humans. These clinical studies have not yet provided convincing evidence that SIRT proteins alleviate morbidity and mortality in patients with cardiovascular diseases. The outcomes of multiple ongoing clinical trials are awaited to define the efficacy of SIRT modulators and SIRT activators in cardiovascular diseases, along with the potential adverse effects of chronic SIRT modulation. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation▿

PubMed Central

Lu, Z.; Altermann, E.; Breidt, F.; Kozyavkin, S.

2010-01-01

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage. PMID:20118355

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

PubMed Central

Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y. C.

2017-01-01

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4). PMID:28737709

Fundamental Characteristics of AAA+ Protein Family Structure and Function

PubMed Central

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

PubMed Central

Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

2011-01-01

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor*

PubMed Central

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J. H.; Risseeuw, Martijn D. P.; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-01-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. PMID:25154561

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

Functional analysis of proteins and protein species using shotgun proteomics and linear mathematics.

PubMed

Hoehenwarter, Wolfgang; Chen, Yanmei; Recuenco-Munoz, Luis; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Covalent post-translational modification of proteins is the primary modulator of protein function in the cell. It greatly expands the functional potential of the proteome compared to the genome. In the past few years shotgun proteomics-based research, where the proteome is digested into peptides prior to mass spectrometric analysis has been prolific in this area. It has determined the kinetics of tens of thousands of sites of covalent modification on an equally large number of proteins under various biological conditions and uncovered a transiently active regulatory network that extends into diverse branches of cellular physiology. In this review, we discuss this work in light of the concept of protein speciation, which emphasizes the entire post-translationally modified molecule and its interactions and not just the modification site as the functional entity. Sometimes, particularly when considering complex multisite modification, all of the modified molecular species involved in the investigated condition, the protein species must be completely resolved for full understanding. We present a mathematical technique that delivers a good approximation for shotgun proteomics data.

Effects of advanced glycation end products on ezrin-dependent functions in LLC-PK1 proximal tubule cells.

PubMed

Bach, Leon A; Gallicchio, Marisa A; McRobert, E Anne; Tikoo, Anjali; Cooper, Mark E

2005-06-01

We have recently shown that advanced glycation products (AGEs) bind to the ERM (ezrin, radixin, moesin) family of proteins. ERM proteins act as cross-linkers between cell membrane proteins and the actin cytoskeleton. They are also involved in signal transduction pathways. They therefore have a critical role in normal cell processes, including modulation of cell shape, adhesion, and motility. We postulate that AGEs may contribute to diabetic complications by disrupting ERM function. In support of this hypothesis, AGEs inhibit ezrin-dependent tubulogenesis of proximal tubule cells. Phosphorylation is an important activating mechanism for ERM proteins, and AGEs inhibit ezrin phosphorylation mediated by the epidermal growth factor receptor.

AKAP-scaffolding proteins and regulation of cardiac physiology

PubMed Central

Mauban, JRH; O'Donnell, M; Warrier, S; Manni, S; Bond, M

2009-01-01

A kinase anchoring proteins (AKAPs) compose a growing list of diverse but functionally related proteins defined by their ability to bind to the regulatory subunit of protein kinase A. AKAPs perform an integral role in the spatiotemporal modulation of a multitude of cellular signaling pathways. This review highlights the extensive role of AKAPs in cardiac excitation/contraction coupling and cardiac physiology. The literature shows that particular AKAPs are involved in cardiac Ca2+ influx, release, re-uptake, and myocyte repolarization. Studies have also suggested roles for AKAPs in cardiac remodeling. Transgenic studies show functional effects of AKAPs, not only in the cardiovascular system, but in other organ systems as well. PMID:19364910

Identification of T1D susceptibility genes within the MHC region by combining protein interaction networks and SNP genotyping data

PubMed Central

Brorsson, C.; Hansen, N. T.; Lage, K.; Bergholdt, R.; Brunak, S.; Pociot, F.

2009-01-01

Aim To develop novel methods for identifying new genes that contribute to the risk of developing type 1 diabetes within the Major Histocompatibility Complex (MHC) region on chromosome 6, independently of the known linkage disequilibrium (LD) between human leucocyte antigen (HLA)-DRB1, -DQA1, -DQB1 genes. Methods We have developed a novel method that combines single nucleotide polymorphism (SNP) genotyping data with protein–protein interaction (ppi) networks to identify disease-associated network modules enriched for proteins encoded from the MHC region. Approximately 2500 SNPs located in the 4 Mb MHC region were analysed in 1000 affected offspring trios generated by the Type 1 Diabetes Genetics Consortium (T1DGC). The most associated SNP in each gene was chosen and genes were mapped to ppi networks for identification of interaction partners. The association testing and resulting interacting protein modules were statistically evaluated using permutation. Results A total of 151 genes could be mapped to nodes within the protein interaction network and their interaction partners were identified. Five protein interaction modules reached statistical significance using this approach. The identified proteins are well known in the pathogenesis of T1D, but the modules also contain additional candidates that have been implicated in β-cell development and diabetic complications. Conclusions The extensive LD within the MHC region makes it important to develop new methods for analysing genotyping data for identification of additional risk genes for T1D. Combining genetic data with knowledge about functional pathways provides new insight into mechanisms underlying T1D. PMID:19143816

Protein conformational disorder and enzyme catalysis.

PubMed

Schulenburg, Cindy; Hilvert, Donald

2013-01-01

Though lacking a well-defined three-dimensional structure, intrinsically unstructured proteins are ubiquitous in nature. These molecules play crucial roles in many cellular processes, especially signaling and regulation. Surprisingly, even enzyme catalysis can tolerate substantial disorder. This observation contravenes conventional wisdom but is relevant to an understanding of how protein dynamics modulates enzyme function. This chapter reviews properties and characteristics of disordered proteins, emphasizing examples of enzymes that lack defined structures, and considers implications of structural disorder for catalytic efficiency and evolution.

Cystic fibrosis transmembrane conductance regulator modulators: precision medicine in cystic fibrosis.

PubMed

Burgener, Elizabeth B; Moss, Richard B

2018-06-01

The aim of this study was to describe the newest development in cystic fibrosis (CF) care, CF transmembrane conductance regulator (CFTR) modulator therapies. Phase II results showing CFTR modulator triple therapies are more effective than current CFTR modulators. CFTR modulator therapy targets the protein defective in CF and boosts its function, but the drug must match mutation pathobiology. Ivacaftor, a CFTR potentiator, was the first modulator approved in 2012, with impressive improvement in lung function and other measures of disease in patients with gating and other residual function mutations (∼10% of CF patients). In 2015, the combination of lumacaftor, a CFTR corrector, and ivacaftor was approved for patients homozygous for the F508del mutation (∼40-50% of the CF population) with positive but less impressive clinical response and 10-20% incidence of intolerance. A next-generation CFTR corrector, tezacaftor, with ivacaftor equally effective and better tolerated than lumacaftor, has also received US Food and Drug Administration approval. Novel CFTR correctors, entering Phase 3 trials in triple modulator combination with tezacaftor-ivacaftor, appear substantially more effective for patients who are homozygous for the F508del mutation and can provide benefit for patients with a single F508del mutation. This offers promise of effective CFTR modulator therapy for nearly 90% of CF patients.

Multiple structure-intrinsic disorder interactions regulate and coordinate Hox protein function

NASA Astrophysics Data System (ADS)

Bondos, Sarah

During animal development, Hox transcription factors determine fate of developing tissues to generate diverse organs and appendages. Hox proteins are famous for their bizarre mutant phenotypes, such as replacing antennae with legs. Clearly, the functions of individual Hox proteins must be distinct and reliable in vivo, or the organism risks malformation or death. However, within the Hox protein family, the DNA-binding homeodomains are highly conserved and the amino acids that contact DNA are nearly invariant. These observations raise the question: How do different Hox proteins correctly identify their distinct target genes using a common DNA binding domain? One possible means to modulate DNA binding is through the influence of the non-homeodomain protein regions, which differ significantly among Hox proteins. However genetic approaches never detected intra-protein interactions, and early biochemical attempts were hindered because the special features of ``intrinsically disordered'' sequences were not appreciated. We propose the first-ever structural model of a Hox protein to explain how specific contacts between distant, intrinsically disordered regions of the protein and the homeodomain regulate DNA binding and coordinate this activity with other Hox molecular functions.

The interaction between thermodynamic stability and buried free cysteines in regulating the functional half-life of fibroblast growth factor-1.

PubMed

Lee, Jihun; Blaber, Michael

2009-10-16

Protein biopharmaceuticals are an important and growing area of human therapeutics; however, the intrinsic property of proteins to adopt alternative conformations (such as during protein unfolding and aggregation) presents numerous challenges, limiting their effective application as biopharmaceuticals. Using fibroblast growth factor-1 as model system, we describe a cooperative interaction between the intrinsic property of thermostability and the reactivity of buried free-cysteine residues that can substantially modulate protein functional half-life. A mutational strategy that combines elimination of buried free cysteines and secondary mutations that enhance thermostability to achieve a substantial gain in functional half-life is described. Furthermore, the implementation of this design strategy utilizing stabilizing mutations within the core region resulted in a mutant protein that is essentially indistinguishable from wild type as regard protein surface and solvent structure, thus minimizing the immunogenic potential of the mutations. This design strategy should be generally applicable to soluble globular proteins containing buried free-cysteine residues.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

PubMed

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO 2 affects plant growth and productivity. CO 2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO 2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO 2 ) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO 2 responses.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

PubMed Central

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses. PMID:28184230

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

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

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest

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

Daviau, Alex; Couture, Jean-Philippe; Blouin, Richard, E-mail: Richard.Blouin@USherbrooke.ca

Highlights: {yields} Role of DLK in cell proliferation. {yields} Modulation of DLK expression during cell cycle progression. {yields} DLK knockdown induces proliferation arrest and senescence. {yields} DLK-depleted cells display loss of cyclin D1 and up-regulation of p21. {yields} DLK participates in cell proliferation by modulating cell cycle regulator expression. -- Abstract: DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, althoughmore » little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.« less

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

PubMed

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

A regulon conserved in monocot and dicot plants defines a functional module in antifungal plant immunity

PubMed Central

Humphry, Matt; Bednarek, Paweł; Kemmerling, Birgit; Koh, Serry; Stein, Mónica; Göbel, Ulrike; Stüber, Kurt; Piślewska-Bednarek, Mariola; Loraine, Ann; Schulze-Lefert, Paul; Somerville, Shauna; Panstruga, Ralph

2010-01-01

At least two components that modulate plant resistance against the fungal powdery mildew disease are ancient and have been conserved since the time of the monocot–dicot split (≈200 Mya). These components are the seven transmembrane domain containing MLO/MLO2 protein and the syntaxin ROR2/PEN1, which act antagonistically and have been identified in the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana, respectively. Additionally, syntaxin-interacting N-ethylmaleimide sensitive factor adaptor protein receptor proteins (VAMP721/722 and SNAP33/34) as well as a myrosinase (PEN2) and an ABC transporter (PEN3) contribute to antifungal resistance in both barley and/or Arabidopsis. Here, we show that these genetically defined defense components share a similar set of coexpressed genes in the two plant species, comprising a statistically significant overrepresentation of gene products involved in regulation of transcription, posttranslational modification, and signaling. Most of the coexpressed Arabidopsis genes possess a common cis-regulatory element that may dictate their coordinated expression. We exploited gene coexpression to uncover numerous components in Arabidopsis involved in antifungal defense. Together, our data provide evidence for an evolutionarily conserved regulon composed of core components and clade/species-specific innovations that functions as a module in plant innate immunity. PMID:21098265

CPI motif interaction is necessary for capping protein function in cells

PubMed Central

Edwards, Marc; McConnell, Patrick; Schafer, Dorothy A.; Cooper, John A.

2015-01-01

Capping protein (CP) has critical roles in actin assembly in vivo and in vitro. CP binds with high affinity to the barbed end of actin filaments, blocking the addition and loss of actin subunits. Heretofore, models for actin assembly in cells generally assumed that CP is constitutively active, diffusing freely to find and cap barbed ends. However, CP can be regulated by binding of the ‘capping protein interaction' (CPI) motif, found in a diverse and otherwise unrelated set of proteins that decreases, but does not abolish, the actin-capping activity of CP and promotes uncapping in biochemical experiments. Here, we report that CP localization and the ability of CP to function in cells requires interaction with a CPI-motif-containing protein. Our discovery shows that cells target and/or modulate the capping activity of CP via CPI motif interactions in order for CP to localize and function in cells. PMID:26412145

Mitogen-activated protein kinase cascades in signaling plant growth and development.

PubMed

Xu, Juan; Zhang, Shuqun

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hypoxia and Redox Signaling on Extracellular Matrix Remodeling: From Mechanisms to Pathological Implications.

PubMed

Labrousse-Arias, David; Martínez-Ruiz, Antonio; Calzada, María J

2017-10-20

The extracellular matrix (ECM) is an essential modulator of cell behavior that influences tissue organization. It has a strong relevance in homeostasis and translational implications for human disease. In addition to ECM structural proteins, matricellular proteins are important regulators of the ECM that are involved in a myriad of different pathologies. Recent Advances: Biochemical studies, animal models, and study of human diseases have contributed to the knowledge of molecular mechanisms involved in remodeling of the ECM, both in homeostasis and disease. Some of them might help in the development of new therapeutic strategies. This review aims to review what is known about some of the most studied matricellular proteins and their regulation by hypoxia and redox signaling, as well as the pathological implications of such regulation. Matricellular proteins have complex regulatory functions and are modulated by hypoxia and redox signaling through diverse mechanisms, in some cases with controversial effects that can be cell or tissue specific and context dependent. Therefore, a better understanding of these regulatory processes would be of great benefit and will open new avenues of considerable therapeutic potential. Characterizing the specific molecular mechanisms that modulate matricellular proteins in pathological processes that involve hypoxia and redox signaling warrants additional consideration to harness the potential therapeutic value of these regulatory proteins. Antioxid. Redox Signal. 27, 802-822.

Regulation of Cytokine Production by the Unfolded Protein Response; Implications for Infection and Autoimmunity

PubMed Central

Smith, Judith A.

2018-01-01

Protein folding in the endoplasmic reticulum (ER) is an essential cell function. To safeguard this process in the face of environmental threats and internal stressors, cells mount an evolutionarily conserved response known as the unfolded protein response (UPR). Invading pathogens induce cellular stress that impacts protein folding, thus the UPR is well situated to sense danger and contribute to immune responses. Cytokines (inflammatory cytokines and interferons) critically mediate host defense against pathogens, but when aberrantly produced, may also drive pathologic inflammation. The UPR influences cytokine production on multiple levels, from stimulation of pattern recognition receptors, to modulation of inflammatory signaling pathways, and the regulation of cytokine transcription factors. This review will focus on the mechanisms underlying cytokine regulation by the UPR, and the repercussions of this relationship for infection and autoimmune/autoinflammatory diseases. Interrogation of viral and bacterial infections has revealed increasing numbers of examples where pathogens induce or modulate the UPR and implicated UPR-modulated cytokines in host response. The flip side of this coin, the UPR/ER stress responses have been increasingly recognized in a variety of autoimmune and inflammatory diseases. Examples include monogenic disorders of ER function, diseases linked to misfolding protein (HLA-B27 and spondyloarthritis), diseases directly implicating UPR and autophagy genes (inflammatory bowel disease), and autoimmune diseases targeting highly secretory cells (e.g., diabetes). Given the burgeoning interest in pharmacologically targeting the UPR, greater discernment is needed regarding how the UPR regulates cytokine production during specific infections and autoimmune processes, and the relative place of this interaction in pathogenesis. PMID:29556237

Investigation of candidate genes for osteoarthritis based on gene expression profiles.

PubMed

Dong, Shuanghai; Xia, Tian; Wang, Lei; Zhao, Qinghua; Tian, Jiwei

2016-12-01

To explore the mechanism of osteoarthritis (OA) and provide valid biological information for further investigation. Gene expression profile of GSE46750 was downloaded from Gene Expression Omnibus database. The Linear Models for Microarray Data (limma) package (Bioconductor project, http://www.bioconductor.org/packages/release/bioc/html/limma.html) was used to identify differentially expressed genes (DEGs) in inflamed OA samples. Gene Ontology function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis of DEGs were performed based on Database for Annotation, Visualization and Integrated Discovery data, and protein-protein interaction (PPI) network was constructed based on the Search Tool for the Retrieval of Interacting Genes/Proteins database. Regulatory network was screened based on Encyclopedia of DNA Elements. Molecular Complex Detection was used for sub-network screening. Two sub-networks with highest node degree were integrated with transcriptional regulatory network and KEGG functional enrichment analysis was processed for 2 modules. In total, 401 up- and 196 down-regulated DEGs were obtained. Up-regulated DEGs were involved in inflammatory response, while down-regulated DEGs were involved in cell cycle. PPI network with 2392 protein interactions was constructed. Moreover, 10 genes including Interleukin 6 (IL6) and Aurora B kinase (AURKB) were found to be outstanding in PPI network. There are 214 up- and 8 down-regulated transcription factor (TF)-target pairs in the TF regulatory network. Module 1 had TFs including SPI1, PRDM1, and FOS, while module 2 contained FOSL1. The nodes in module 1 were enriched in chemokine signaling pathway, while the nodes in module 2 were mainly enriched in cell cycle. The screened DEGs including IL6, AGT, and AURKB might be potential biomarkers for gene therapy for OA by being regulated by TFs such as FOS and SPI1, and participating in the cell cycle and cytokine-cytokine receptor interaction pathway. Copyright © 2016 Turkish Association of Orthopaedics and Traumatology. Production and hosting by Elsevier B.V. All rights reserved.

Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

PubMed

Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

2014-01-01

The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. © 2013 Anatomical Society.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

A Variable Polyglutamine Repeat Affects Subcellular Localization and Regulatory Activity of a Populus ANGUSTIFOLIA Protein.

PubMed

Bryan, Anthony C; Zhang, Jin; Guo, Jianjun; Ranjan, Priya; Singan, Vasanth; Barry, Kerrie; Schmutz, Jeremy; Weighill, Deborah; Jacobson, Daniel; Jawdy, Sara; Tuskan, Gerald A; Chen, Jin-Gui; Muchero, Wellington

2018-06-08

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA ( PtAN1 ) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates PtAN1 function by altering subcellular localization. Copyright © 2018, G3: Genes, Genomes, Genetics.

A Glutathione Peroxidase, Intracellular Peptidases and the TOR Complexes Regulate Peptide Transporter PEPT-1 in C. elegans

PubMed Central

Benner, Jacqueline; Daniel, Hannelore; Spanier, Britta

2011-01-01

The intestinal peptide transporter PEPT-1 in Caenorhabditis elegans is a rheogenic H+-dependent carrier responsible for the absorption of di- and tripeptides. Transporter-deficient pept-1(lg601) worms are characterized by impairments in growth, development and reproduction and develop a severe obesity like phenotype. The transport function of PEPT-1 as well as the influx of free fatty acids was shown to be dependent on the membrane potential and on the intracellular pH homeostasis, both of which are regulated by the sodium-proton exchanger NHX-2. Since many membrane proteins commonly function as complexes, there could be proteins that possibly modulate PEPT-1 expression and function. A systematic RNAi screening of 162 genes that are exclusively expressed in the intestine combined with a functional transport assay revealed four genes with homologues existing in mammals as predicted PEPT-1 modulators. While silencing of a glutathione peroxidase surprisingly caused an increase in PEPT-1 transport function, silencing of the ER to Golgi cargo transport protein and of two cytosolic peptidases reduced PEPT-1 transport activity and this even corresponded with lower PEPT-1 protein levels. These modifications of PEPT-1 function by gene silencing of homologous genes were also found to be conserved in the human epithelial cell line Caco-2/TC7 cells. Peptidase inhibition, amino acid supplementation and RNAi silencing of targets of rapamycin (TOR) components in C. elegans supports evidence that intracellular peptide hydrolysis and amino acid concentration are a part of a sensing system that controls PEPT-1 expression and function and that involves the TOR complexes TORC1 and TORC2. PMID:21980510

Evidence for dynamically organized modularity in the yeast protein-protein interaction network

NASA Astrophysics Data System (ADS)

Han, Jing-Dong J.; Bertin, Nicolas; Hao, Tong; Goldberg, Debra S.; Berriz, Gabriel F.; Zhang, Lan V.; Dupuy, Denis; Walhout, Albertha J. M.; Cusick, Michael E.; Roth, Frederick P.; Vidal, Marc

2004-07-01

In apparently scale-free protein-protein interaction networks, or `interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the `hubs', interact with many partners. Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs. Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space. We uncovered two types of hub: `party' hubs, which interact with most of their partners simultaneously, and `date' hubs, which bind their different partners at different times or locations. Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes-or modules -to each other, whereas party hubs function inside modules.

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

PubMed

Rues, Ralf-Bernhardt; Gräwe, Alexander; Henrich, Erik; Bernhard, Frank

2017-01-01

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

A multitasking Argonaute: exploring the many facets of C. elegans CSR-1.

PubMed

Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

2013-12-01

While initial studies of small RNA-mediated gene regulatory pathways focused on the cytoplasmic functions of such pathways, identifying roles for Argonaute/small RNA pathways in modulating chromatin and organizing the genome has become a topic of intense research in recent years. Nuclear regulatory mechanisms for Argonaute/small RNA pathways appear to be widespread, in organisms ranging from plants to fission yeast, Caenorhabditis elegans to humans. As the effectors of small RNA-mediated gene regulatory pathways, Argonaute proteins guide the chromatin-directed activities of these pathways. Of particular interest is the C. elegans Argonaute, chromosome segregation and RNAi deficient (CSR-1), which has been implicated in such diverse functions as organizing the holocentromeres of worm chromosomes, modulating germline chromatin, protecting the genome from foreign nucleic acid, regulating histone levels, executing RNAi, and inhibiting translation in conjunction with Pumilio proteins. CSR-1 interacts with small RNAs known as 22G-RNAs, which have complementarity to 25 % of the protein coding genes. This peculiar Argonaute is the only essential C. elegans Argonaute out of 24 family members in total. Here, we summarize the current understanding of CSR-1 functions in the worm, with emphasis on the chromatin-directed activities of this ever-intriguing Argonaute.

Characterization of strychnine-sensitive glycine receptor in the intact frog retina: modulation by protein kinases.

PubMed

Salceda, Rocío; Aguirre-Ramirez, Marisela

2005-03-01

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

Strong G-Protein-Mediated Inhibition of Sodium Channels.

PubMed

Mattheisen, Glynis B; Tsintsadze, Timur; Smith, Stephen M

2018-05-29

Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability. Published by Elsevier Inc.

FMRP acts as a key messenger for dopamine modulation in the forebrain.

PubMed

Wang, Hansen; Wu, Long-Jun; Kim, Susan S; Lee, Frank J S; Gong, Bo; Toyoda, Hiroki; Ren, Ming; Shang, Yu-Ze; Xu, Hui; Liu, Fang; Zhao, Ming-Gao; Zhuo, Min

2008-08-28

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. Here, we report that FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1(-/-) prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1(-/-) mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1(-/-) neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1(-/-) mice. Our study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

PubMed

Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

The additive effects of atorvastatin and insulin on renal function and renal organic anion transporter 3 function in diabetic rats.

PubMed

Thongnak, Laongdao; Pongchaidecha, Anchalee; Jaikumkao, Krit; Chatsudthipong, Varanuj; Chattipakorn, Nipon; Lungkaphin, Anusorn

2017-10-19

Hyperglycemia-induced oxidative stress is usually found in diabetic condition. 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, statins, are widely used as cholesterol-lowering medication with several "pleiotropic" effects in diabetic patients. This study aims to evaluate whether the protective effects of atorvastatin and insulin on renal function and renal organic anion transporter 3 (Oat3) function involve the modulation of oxidative stress and pancreatic function in type 1 diabetic rats. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin (50 mg/kg BW). Atorvastatin and insulin as single or combined treatment were given for 4 weeks after diabetic condition had been confirmed. Diabetic rats demonstrated renal function and renal Oat3 function impairment with an increased MDA level and decreased SOD protein expression concomitant with stimulation of renal Nrf2 and HO-1 protein expression. Insulin plus atorvastatin (combined) treatment effectively restored renal function as well as renal Oat3 function which correlated with the decrease in hyperglycemia and oxidative stress. Moreover, pancreatic inflammation and apoptosis in diabetic rats were ameliorated by the combined drugs treatment. Therefore, atorvastatin plus insulin seems to exert the additive effect in improving renal functionby alleviating hyperglycemiaand the modulation of oxidative stress, inflammation and apoptosis.

Co-expression network with protein-protein interaction and transcription regulation in malaria parasite Plasmodium falciparum.

PubMed

Yu, Fu-Dong; Yang, Shao-You; Li, Yuan-Yuan; Hu, Wei

2013-04-10

Malaria continues to be one of the most severe global infectious diseases, as a major threat to human health and economic development. Network-based biological analysis is a promising approach to uncover key genes and biological processes from a network viewpoint, which could not be recognized from individual gene-based signatures. We integrated gene co-expression profile with protein-protein interaction and transcriptional regulation information to construct a comprehensive gene co-expression network of Plasmodium falciparum. Based on this network, we identified 10 core modules by using ICE (Iterative Clique Enumeration) algorithm, which were essential for malaria parasite development in intraerythrocytic developmental cycle (IDC) stages. In each module, all genes were highly correlated probably due to co-regulation or formation of a protein complex. Some of these genes were recognized to be differentially coexpressed among three close-by IDC stages. The gene of prpf8 (PFD0265w) encoding pre-mRNA processing splicing factor 8 product was identified as DCGs (differentially co-expressed genes) among IDC stages, although this gene function was seldom reported in previous researches. Integrating the species-specific gene prediction and differential co-expression gene detection, we found some modules could perform species-specific functions according to some of genes in these modules were species-specific genes, like the module 10. Furthermore, in order to reveal the underlying mechanisms of the erythrocyte invasion by P. falciparum, Steiner Tree algorithm was employed to identify the invasion subnetwork from our gene co-expression network. The subnetwork-based analysis indicated that some important Plasmodium parasite specific genes could corporate with each other and be co-regulated during the parasite invasion process, which including a head-to-head gene pair of PfRH2a (PF13_0198) and PfRH2b (MAL13P1.176). This study based on gene co-expression network could shed new insights on the mechanisms of pathogenesis, even virulence and P. falciparum development. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

PubMed

Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

2018-01-01

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-02-01

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

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

PubMed Central

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

2013-01-01

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

SIRT1 is a Highly Networked Protein That Mediates the Adaptation to Chronic Physiological Stress

PubMed Central

Clark-Knowles, Katherine V.; Caron, Annabelle Z.; Gray, Douglas A.

2013-01-01

SIRT1 is a NAD+-dependent protein deacetylase that has a very large number of established protein substrates and an equally impressive list of biological functions thought to be regulated by its activity. Perhaps as notable is the remarkable number of points of conflict concerning the role of SIRT1 in biological processes. For example, evidence exists suggesting that SIRT1 is a tumor suppressor, is an oncogene, or has no effect on oncogenesis. Similarly, SIRT1 is variably reported to induce, inhibit, or have no effect on autophagy. We believe that the resolution of many conflicting results is possible by considering recent reports indicating that SIRT1 is an important hub interacting with a complex network of proteins that collectively regulate a wide variety of biological processes including cancer and autophagy. A number of the interacting proteins are themselves hubs that, like SIRT1, utilize intrinsically disordered regions for their promiscuous interactions. Many studies investigating SIRT1 function have been carried out on cell lines carrying undetermined numbers of alterations to the proteins comprising the SIRT1 network or on inbred mouse strains carrying fixed mutations affecting some of these proteins. Thus, the effects of modulating SIRT1 amount and/or activity are importantly determined by the genetic background of the cell (or the inbred strain of mice), and the effects attributed to SIRT1 are synthetic with the background of mutations and epigenetic differences between cells and organisms. Work on mice carrying alterations to the Sirt1 gene suggests that the network in which SIRT1 functions plays an important role in mediating physiological adaptation to various sources of chronic stress such as calorie restriction and calorie overload. Whether the catalytic activity of SIRT1 and the nuclear concentration of the co-factor, NAD+, are responsible for modulating this activity remains to be determined. However, the effect of modulating SIRT1 activity must be interpreted in the context of the cell or tissue under investigation. Indeed, for SIRT1, we argue that context is everything. PMID:24020004

A-Kinase Anchoring Proteins That Regulate Cardiac Remodeling

PubMed Central

Carnegie, Graeme K.; Burmeister, Brian T.

2012-01-01

In response to injury or stress, the adult heart undergoes maladaptive changes, collectively defined as pathological cardiac remodeling. Here, we focus on the role of A-kinase anchoring proteins (AKAPs) in 3 main areas associated with cardiac remodeling and the progression of heart failure: excitation–contraction coupling, sarcomeric regulation, and induction of pathological hypertrophy. AKAPs are a diverse family of scaffold proteins that form multi-protein complexes, integrating cAMP signaling with protein kinases, phosphatases, and other effector proteins. Many AKAPs have been characterized in the heart, where they play a critical role in modulating cardiac function. PMID:22075671

Molecular Dynamics Simulations of Protein-Ligand Complexes in Near Physiological Conditions

NASA Astrophysics Data System (ADS)

Wambo, Thierry Oscar

Proteins are important molecules for their key functions. However, under certain circumstances, the function of these proteins needs to be regulated to keep us healthy. Ligands are small molecules often used to modulate the function of proteins. The binding affinity is a quantitative measure of how strong the ligand will modulate the function of the protein: a strong binding affinity will highly impact the performance of the protein. It becomes clear that it is critical to have appropriate techniques to accurately compute the binding affinity. The most difficult task in computer simulations is how to efficiently sample the space spanned by the ligand during the binding process. In this work, we have developed some schemes to compute the binding affinity of a ligand to a protein, and of a metal ion to a protein. Application of these techniques to some complexes yield results in agreement with experimental values. These methods are a brute force approach and make no assumption other than that the complexes are governed by the force field used. Specifically, we computed the free energy of binding between (1) human carbonic anhydrase II and the drug acetazolamide (hcaII-AZM), (2) human carbonic anhydrase II and the zinc ion (hcaII-Zinc), and (3) beta-lactoglobulin and five fatty acids complexes (BLG-FAs). We found the following free energies of binding in unit of kcal/mol: -12.96 +/-2.44 (-15.74) for hcaII-Zinc complex, -5.76+/-0.76 (-5.57) for BLG-OCA , -4.44+/-1.08 (-5.22) for BLG-DKA,-6.89+/-1.25 (-7.24) for BLG-DAO, -8.57+/-0.82 (-8.14) for BLG-MYR, -8.99+/-0.87 (-8.72) for BLG-PLM, and -11.87+/-1.8 (-10.8) for hcaII-AZM. The values inside the parentheses are experimental results. The simulations and quantitative analysis of each system provide interesting insights into the interactions between each entity and helps us to better understand the dynamics of these systems.

The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants.

PubMed

Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

2014-01-01

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

PubMed Central

Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

2014-01-01

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation. PMID:24600465

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

PubMed

Maher, Christina M; Thomas, Jeffrey D; Haas, Derick A; Longen, Charles G; Oyer, Halley M; Tong, Jane Y; Kim, Felix J

2018-02-01

Emerging evidence suggests that Sigma1 ( SIGMAR1 , also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR . ©2017 American Association for Cancer Research.

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

PubMed Central

2009-01-01

The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold. PMID:19930717

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

PubMed Central

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-01-01

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

PubMed Central

Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

2015-01-01

Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

Novel role of phosphorylation in Fe–S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1

PubMed Central

Brown, Nina M.; Anderson, Sheila A.; Steffen, Daniel W.; Carpenter, Tami B.; Kennedy, M. Claire; Walden, William E.; Eisenstein, Richard S.

1998-01-01

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe–4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe–4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe–S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe–4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression. PMID:9860952

Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro.

PubMed

Cartocci, Veronica; Segatto, Marco; Di Tunno, Ilenia; Leone, Stefano; Pfrieger, Frank W; Pallottini, Valentina

2016-09-01

During differentiation, neurons acquire their typical shape and functional properties. At present, it is unclear, whether this important developmental step involves metabolic changes. Here, we studied the contribution of the mevalonate (MVA) pathway to neuronal differentiation using the mouse neuroblastoma cell line N1E-115 as experimental model. Our results show that during differentiation, the activity of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR), a key enzyme of MVA pathway, and the level of Low Density Lipoprotein receptor (LDLr) decrease, whereas the level of LDLr-related protein-1 (LRP1) and the dimerization of Scavanger Receptor B1 (SRB-1) rise. Pharmacologic inhibition of HMGR by simvastatin accelerated neuronal differentiation by modulating geranylated proteins. Collectively, our data suggest that during neuronal differentiation, the activity of the MVA pathway decreases and we postulate that any interference with this process impacts neuronal morphology and function. Therefore, the MVA pathway appears as an attractive pharmacological target to modulate neurological and metabolic symptoms of developmental neuropathologies. J. Cell. Biochem. 117: 2036-2044, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The sigma-1 receptor chaperone as an inter-organelle signaling modulator

PubMed Central

Su, Tsung-Ping; Hayashi, Teruo; Maurice, Tangui; Buch, Shilpa; Ruoho, Arnold E.

2010-01-01

Inter-organelle signaling plays important roles in many physiological functions. Endoplasmic reticulum (ER)-mitochondrion signaling affects intra-mitochondrial calcium (Ca2+) homeostasis and cellular bioenergetics. ER-nucleus signaling attenuates ER stress. ER-plasma membrane signaling regulates cytosolic Ca2+ homeostasis, and ER-mitochondrion-plasma membrane signaling regulates hippocampal dendritic spine formation. Here we propose that the sigma-1 receptor (Sig-1R), an ER chaperone protein, acts as an inter-organelle signaling modulator. Sig-1Rs normally reside at the ER-mitochondrion contact called the MAM (mitochondrion-associated ER membrane), where Sig-1Rs regulate ER-mitochondrion signaling and the ER-nucleus cross-talk. When cells are stimulated by ligands or undergo prolonged stress, Sig-1Rs translocate from the MAM to the ER reticular network and plasmalemma/plasma membrane to regulate a variety of functional proteins, including ion channels, receptors, and kinases. Thus, the Sig-1R serves as an inter-organelle signaling modulator locally at the MAM and remotely at the plasmalemma/plasma membrane. Many pharmacological/physiological effects of Sig-1Rs may relate to this unique action of Sig-1Rs. PMID:20869780

BAG3 down-modulation sensitizes HPV18(+) HeLa cells to PEITC-induced apoptosis and restores p53.

PubMed

Cotugno, Roberta; Basile, Anna; Romano, Elena; Gallotta, Dario; Belisario, Maria Antonietta

2014-11-28

BAG3 is a multi-functional component of tumor cell pro-survival machinery, and its biological functions have been largely associated to proteasome system. Here, we show that BAG3 down-modulation resulted in reduced cell viability and enhanced PEITC-induced apoptosis largely more extensively in HeLa (HPV18(+)) rather than in C33A (HPV(-)) cervical carcinoma cell lines. Moreover, we demonstrate that BAG3 suppression led to a decrease of viral E6 oncoprotein and a concomitant recovery of p53 tumor suppressor, the best recognized target of E6 for proteasome degradation. E6 and p53 expression were modulated at protein level, since their respective mRNAs were unaffected. Taken together our findings reveal a novel role for BAG3 as host protein contributing to HPV18 E6-activated pro-survival strategies, and suggest a possible relevance of its expression levels in drug/radiotherapy-resistance of HPV18-bearing cervical carcinomas. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

PubMed Central

Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

2013-01-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function.

PubMed

Back, Steven H; Adapala, Naga Suresh; Barbe, Mary F; Carpino, Nick C; Tsygankov, Alexander Y; Sanjay, Archana

2013-04-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

PubMed

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

Evolutionary conservation of Ebola virus proteins predicts important functions at residue level.

PubMed

Arslan, Ahmed; van Noort, Vera

2017-01-15

The recent outbreak of Ebola virus disease (EVD) resulted in a large number of human deaths. Due to this devastation, the Ebola virus has attracted renewed interest as model for virus evolution. Recent literature on Ebola virus (EBOV) has contributed substantially to our understanding of the underlying genetics and its scope with reference to the 2014 outbreak. But no study yet, has focused on the conservation patterns of EBOV proteins. We analyzed the evolution of functional regions of EBOV and highlight the function of conserved residues in protein activities. We apply an array of computational tools to dissect the functions of EBOV proteins in detail: (i) protein sequence conservation, (ii) protein-protein interactome analysis, (iii) structural modeling and (iv) kinase prediction. Our results suggest the presence of novel post-translational modifications in EBOV proteins and their role in the modulation of protein functions and protein interactions. Moreover, on the basis of the presence of ATM recognition motifs in all EBOV proteins we postulate a role of DNA damage response pathways and ATM kinase in EVD. The ATM kinase is put forward, for further evaluation, as novel potential therapeutic target. http://www.biw.kuleuven.be/CSB/EBOV-PTMs CONTACT: vera.vannoort@biw.kuleuven.beSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Detection of protein complex from protein-protein interaction network using Markov clustering

NASA Astrophysics Data System (ADS)

Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

2017-05-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

Alternative ways of modulating JAK-STAT pathway

PubMed Central

2012-01-01

Most attempts to develop inhibitors of STAT transcription factors target either activating phosphorylation of tyrosine residue or SH2 domains. However, all six domains of STATs are highly conserved between the species and play important roles in the function of this family of transcription factors. STATs are involved in numerous protein-protein interactions that are likely to regulate and fine tune transcriptional activity. Targeting these interactions can provide plentiful opportunities for the discovery of novel drug candidates and powerful chemical biology tools. Using N-terminal domains as an example we describe alternative rational approaches to the development of modulators of JAK-STAT signaling. PMID:24058784

Modifications of Glycans: Biological Significance and Therapeutic Opportunities

PubMed Central

Muthana, Saddam M.; Campbell, Christopher; Gildersleeve, Jeffrey C.

2012-01-01

Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate’s overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as postglycosylational modifications. PMID:22195988

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

PubMed Central

Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

2013-01-01

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

Heteroreceptors Modulating CGRP Release at Neurovascular Junction: Potential Therapeutic Implications on Some Vascular-Related Diseases.

PubMed

González-Hernández, Abimael; Marichal-Cancino, Bruno A; Lozano-Cuenca, Jair; López-Canales, Jorge S; Muñoz-Islas, Enriqueta; Ramírez-Rosas, Martha B; Villalón, Carlos M

2016-01-01

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid neuropeptide belonging to the calcitonin gene peptide superfamily. CGRP is a potent vasodilator with potential therapeutic usefulness for treating vascular-related disease. This peptide is primarily located on C- and A δ -fibers, which have extensive perivascular presence and a dual sensory-efferent function. Although CGRP has two major isoforms ( α -CGRP and β -CGRP), the α -CGRP is the isoform related to vascular actions. Release of CGRP from afferent perivascular nerve terminals has been shown to result in vasodilatation, an effect mediated by at least one receptor (the CGRP receptor). This receptor is an atypical G-protein coupled receptor (GPCR) composed of three functional proteins: (i) the calcitonin receptor-like receptor (CRLR; a seven-transmembrane protein), (ii) the activity-modifying protein type 1 (RAMP1), and (iii) a receptor component protein (RCP). Although under physiological conditions, CGRP seems not to play an important role in vascular tone regulation, this peptide has been strongly related as a key player in migraine and other vascular-related disorders (e.g., hypertension and preeclampsia). The present review aims at providing an overview on the role of sensory fibers and CGRP release on the modulation of vascular tone.

Heteroreceptors Modulating CGRP Release at Neurovascular Junction: Potential Therapeutic Implications on Some Vascular-Related Diseases

PubMed Central

Marichal-Cancino, Bruno A.; Lozano-Cuenca, Jair; López-Canales, Jorge S.; Muñoz-Islas, Enriqueta; Ramírez-Rosas, Martha B.; Villalón, Carlos M.

2016-01-01

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid neuropeptide belonging to the calcitonin gene peptide superfamily. CGRP is a potent vasodilator with potential therapeutic usefulness for treating vascular-related disease. This peptide is primarily located on C- and Aδ-fibers, which have extensive perivascular presence and a dual sensory-efferent function. Although CGRP has two major isoforms (α-CGRP and β-CGRP), the α-CGRP is the isoform related to vascular actions. Release of CGRP from afferent perivascular nerve terminals has been shown to result in vasodilatation, an effect mediated by at least one receptor (the CGRP receptor). This receptor is an atypical G-protein coupled receptor (GPCR) composed of three functional proteins: (i) the calcitonin receptor-like receptor (CRLR; a seven-transmembrane protein), (ii) the activity-modifying protein type 1 (RAMP1), and (iii) a receptor component protein (RCP). Although under physiological conditions, CGRP seems not to play an important role in vascular tone regulation, this peptide has been strongly related as a key player in migraine and other vascular-related disorders (e.g., hypertension and preeclampsia). The present review aims at providing an overview on the role of sensory fibers and CGRP release on the modulation of vascular tone. PMID:28116293

Abnormally high expression of POLD1, MCM2, and PLK4 promotes relapse of acute lymphoblastic leukemia.

PubMed

Li, Sheng; Wang, Chengzhong; Wang, Weikai; Liu, Weidong; Zhang, Guiqin

2018-05-01

This study aimed to explore the underlying mechanism of relapsed acute lymphoblastic leukemia (ALL).Datasets of GSE28460 and GSE18497 were downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) between diagnostic and relapsed ALL samples were identified using Limma package in R, and a Venn diagram was drawn. Next, functional enrichment analyses of co-regulated DEGs were performed. Based on the String database, protein-protein interaction network and module analyses were also conducted. Moreover, transcription factors and miRNAs targeting co-regulated DEGs were predicted using the WebGestalt online tool.A total of 71 co-regulated DEGs were identified, including 56 co-upregulated genes and 15 co-downregulated genes. Functional enrichment analyses showed that upregulated DEGs were significantly enriched in the cell cycle, and DNA replication, and repair related pathways. POLD1, MCM2, and PLK4 were hub proteins in both protein-protein interaction network and module, and might be potential targets of E2F. Additionally, POLD1 and MCM2 were found to be regulated by miR-520H via E2F1.High expression of POLD1, MCM2, and PLK4 might play positive roles in the recurrence of ALL, and could serve as potential therapeutic targets for the treatment of relapsed ALL.

An m6A-YTH Module Controls Developmental Timing and Morphogenesis in Arabidopsis.

PubMed

Arribas-Hernández, Laura; Bressendorff, Simon; Hansen, Mathias Henning; Poulsen, Christian; Erdmann, Susanne; Brodersen, Peter

2018-04-11

Methylation of N6-adenosine (m6A) in mRNA is an important post-transcriptional gene regulatory mechanism in eukaryotes. m6A provides a binding site for effector proteins ("readers") that influence pre-mRNA splicing, mRNA degradation or translational efficiency. YT521-B homology (YTH) domain proteins are important m6A readers with established functions in animals. Plants contain more YTH domain proteins than other eukaryotes, but their biological importance remains unknown. Here, we show that the cytoplasmic Arabidopsis thaliana YTH domain proteins EVOLUTIONARILY CONSERVED C-TERMINAL REGION2/3 (ECT2/3) are required for the correct timing of leaf formation and for normal leaf morphology. These functions depend fully on intact m6A binding sites of ECT2 and ECT3, indicating that they function as m6A readers. Mutation of the close ECT2 homolog, ECT4, enhances the delayed leaf emergence and leaf morphology defects of ect2/ect3 mutants, and all three ECT proteins are expressed at leaf formation sites in the shoot apex of young seedlings and in the division zone of developing leaves. ECT2 and ECT3 are also highly expressed at early stages of trichome development and are required for trichome morphology, as previously reported for m6A itself. Overall, our study establishes the relevance of a cytoplasmic m6A-YTH regulatory module in the timing and execution of plant organogenesis. © 2018 American Society of Plant Biologists. All rights reserved.

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

PubMed Central

Romberg, Christin F; Beqollari, Donald; Meza, Ulises; Bannister, Roger A

2014-01-01

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca2+ channel, and (3) voltage-sensor for slow depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry. Our observations provide the first evidence of modulation of this enigmatic Ca2+ entry pathway peculiar to skeletal muscle. PMID:24476902

Muscle Contraction Regulates BDNF/TrkB Signaling to Modulate Synaptic Function through Presynaptic cPKCα and cPKCβI.

PubMed

Hurtado, Erica; Cilleros, Víctor; Nadal, Laura; Simó, Anna; Obis, Teresa; Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Halievski, Katherine; Jordan, Cynthia L; Lanuza, Maria A; Tomàs, Josep

2017-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression in vivo at the neuromuscular junction (NMJ). Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function.

Muscle Contraction Regulates BDNF/TrkB Signaling to Modulate Synaptic Function through Presynaptic cPKCα and cPKCβI

PubMed Central

Hurtado, Erica; Cilleros, Víctor; Nadal, Laura; Simó, Anna; Obis, Teresa; Garcia, Neus; Santafé, Manel M.; Tomàs, Marta; Halievski, Katherine; Jordan, Cynthia L.; Lanuza, Maria A.; Tomàs, Josep

2017-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression in vivo at the neuromuscular junction (NMJ). Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function. PMID:28572757

Genetic Polymorphism in Extracellular Regulators of Wnt Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Seo, Eun-Min; Nam, Ju-Suk

2015-01-01

The Wnt signaling pathway is mediated by a family of secreted glycoproteins through canonical and noncanonical mechanism. The signaling pathways are regulated by various modulators, which are classified into two classes on the basis of their interaction with either Wnt or its receptors. Secreted frizzled-related proteins (sFRPs) are the member of class that binds to Wnt protein and antagonizes Wnt signaling pathway. The other class consists of Dickkopf (DKK) proteins family that binds to Wnt receptor complex. The present review discusses the disease related association of various polymorphisms in Wnt signaling modulators. Furthermore, this review also highlights that some of the sFRPs and DKKs are unable to act as an antagonist for Wnt signaling pathway and thus their function needs to be explored more extensively. PMID:25945348

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins

Modulation of electronic structures of bases through DNA recognition of protein.

PubMed

Hagiwara, Yohsuke; Kino, Hiori; Tateno, Masaru

2010-04-21

The effects of environmental structures on the electronic states of functional regions in a fully solvated DNA·protein complex were investigated using combined ab initio quantum mechanics/molecular mechanics calculations. A complex of a transcriptional factor, PU.1, and the target DNA was used for the calculations. The effects of solvent on the energies of molecular orbitals (MOs) of some DNA bases strongly correlate with the magnitude of masking of the DNA bases from the solvent by the protein. In the complex, PU.1 causes a variation in the magnitude among DNA bases by means of directly recognizing the DNA bases through hydrogen bonds and inducing structural changes of the DNA structure from the canonical one. Thus, the strong correlation found in this study is the first evidence showing the close quantitative relationship between recognition modes of DNA bases and the energy levels of the corresponding MOs. Thus, it has been revealed that the electronic state of each base is highly regulated and organized by the DNA recognition of the protein. Other biological macromolecular systems can be expected to also possess similar modulation mechanisms, suggesting that this finding provides a novel basis for the understanding for the regulation functions of biological macromolecular systems.

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

PubMed

Matheny, Sharon A; Chen, Chiyuan; Kortum, Robert L; Razidlo, Gina L; Lewis, Robert E; White, Michael A

2004-01-15

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

Functional diversification and specialization of cytosolic 70-kDa heat shock proteins.

PubMed

McCallister, Chelsea; Siracusa, Matthew C; Shirazi, Farzaneh; Chalkia, Dimitra; Nikolaidis, Nikolas

2015-03-20

A fundamental question in molecular evolution is how protein functional differentiation alters the ability of cells and organisms to cope with stress and survive. To answer this question we used two paralogous Hsp70s from mouse and explored whether these highly similar cytosolic molecular chaperones, which apart their temporal expression have been considered functionally interchangeable, are differentiated with respect to their lipid-binding function. We demonstrate that the two proteins bind to diverse lipids with different affinities and therefore are functionally specialized. The observed lipid-binding patterns may be related with the ability of both Hsp70s to induce cell death by binding to a particular plasma-membrane lipid, and the potential of only one of them to promote cell survival by binding to a specific lysosomal-membrane lipid. These observations reveal that two seemingly identical proteins differentially modulate cellular adaptation and survival by having acquired specialized functions via sequence divergence. Therefore, this study provides an evolutionary paradigm, where promiscuity, specificity, sub- and neo-functionalization orchestrate one of the most conserved systems in nature, the cellular stress-response.

Identification of the proteins related to SET-mediated hepatic cytotoxicity of trichloroethylene by proteomic analysis.

PubMed

Ren, Xiaohu; Yang, Xifei; Hong, Wen-Xu; Huang, Peiwu; Wang, Yong; Liu, Wei; Ye, Jinbo; Huang, Haiyan; Huang, Xinfeng; Shen, Liming; Yang, Linqing; Zhuang, Zhixiong; Liu, Jianjun

2014-05-16

Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm.

PubMed

Yu, Isseki; Mori, Takaharu; Ando, Tadashi; Harada, Ryuhei; Jung, Jaewoon; Sugita, Yuji; Feig, Michael

2016-11-01

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology.

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

PubMed Central

Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

2013-01-01

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

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

Liu, Xiyuan; An, Byoung Ha; Kim, Min Jung

2014-09-26

Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1more » (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.« less

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

Characterization of Steroid Receptor RNA Activator Protein Function in Modulating the Estrogen Signaling Pathway

DTIC Science & Technology

2007-05-01

Signaling Pathway 5b. GRANT NUMBER W81XWH-05-1-0245 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Yi Yan 5e. TASK NUMBER...negative control to identify proteins non-specifically precipitated. Tandem mass spectrometric analysis of immunoprecipitated samples identified a...immunoprecipitated sample and negative control. It is important to note that, SRAP was present among the remaining specifically precipitated 87 proteins. Using the

Necdin interacts with the Msx2 homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells.

PubMed

Kuwajima, Takaaki; Taniura, Hideo; Nishimura, Isao; Yoshikawa, Kazuaki

2004-09-24

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

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

Neurotrophin-3 Enhances the Synaptic Organizing Function of TrkC-Protein Tyrosine Phosphatase σ in Rat Hippocampal Neurons.

PubMed

Ammendrup-Johnsen, Ina; Naito, Yusuke; Craig, Ann Marie; Takahashi, Hideto

2015-09-09

Neurotrophin-3 (NT-3) and its high-affinity receptor TrkC play crucial trophic roles in neuronal differentiation, axon outgrowth, and synapse development and plasticity in the nervous system. We demonstrated previously that postsynaptic TrkC functions as a glutamatergic synapse-inducing (synaptogenic) cell adhesion molecule trans-interacting with presynaptic protein tyrosine phosphatase σ (PTPσ). Given that NT-3 and PTPσ bind distinct domains of the TrkC extracellular region, here we tested the hypothesis that NT-3 modulates TrkC/PTPσ binding and synaptogenic activity. NT-3 enhanced PTPσ binding to cell surface-expressed TrkC and facilitated the presynapse-inducing activity of TrkC in rat hippocampal neurons. Imaging of recycling presynaptic vesicles combined with TrkC knockdown and rescue approaches demonstrated that NT-3 rapidly potentiates presynaptic function via binding endogenous postsynaptic TrkC in a tyrosine kinase-independent manner. Thus, NT-3 positively modulates the TrkC-PTPσ complex for glutamatergic presynaptic assembly and function independently from TrkC kinase activation. Our findings provide new insight into synaptic roles of neurotrophin signaling and mechanisms controlling synaptic organizing complexes. Significance statement: Although many synaptogenic adhesion complexes have been identified in recent years, little is known about modulatory mechanisms. Here, we demonstrate a novel role of neurotrophin-3 in synaptic assembly and function as a positive modulator of the TrkC-protein tyrosine phosphatase σ complex. This study provides new insight into the involvement of neurotrophin signaling in synapse development and plasticity, presenting a molecular mechanism that may underlie previous observations of short- and long-term enhancement of presynaptic function by neurotrophin. Given the links of synaptogenic adhesion molecules to autism and schizophrenia, this study might also contribute to a better understanding of the pathogenesis of these disorders and provide a new direction for ameliorating imbalances in synaptic signaling networks. Copyright © 2015 the authors 0270-6474/15/3512425-07$15.00/0.

BIOLOGICAL AND BIOPHYSICAL PROPERTIES OF VASCULAR CONNEXIN CHANNELS

PubMed Central

Johnstone, Scott; Isakson, Brant; Locke, Darren

2010-01-01

Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell type-independent and cell type-specific transcription factors, posttranslational modification and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this review in the physiological and pathophysiological context of vessel function. PMID:19815177

A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2

PubMed Central

Hambly, Emma; Tétart, Francoise; Desplats, Carine; Wilson, William H.; Krisch, Henry M.; Mann, Nicholas H.

2001-01-01

Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules. PMID:11553768

Evolution of sparsity and modularity in a model of protein allostery

NASA Astrophysics Data System (ADS)

Hemery, Mathieu; Rivoire, Olivier

2015-04-01

The sequence of a protein is not only constrained by its physical and biochemical properties under current selection, but also by features of its past evolutionary history. Understanding the extent and the form that these evolutionary constraints may take is important to interpret the information in protein sequences. To study this problem, we introduce a simple but physical model of protein evolution where selection targets allostery, the functional coupling of distal sites on protein surfaces. This model shows how the geometrical organization of couplings between amino acids within a protein structure can depend crucially on its evolutionary history. In particular, two scenarios are found to generate a spatial concentration of functional constraints: high mutation rates and fluctuating selective pressures. This second scenario offers a plausible explanation for the high tolerance of natural proteins to mutations and for the spatial organization of their least tolerant amino acids, as revealed by sequence analysis and mutagenesis experiments. It also implies a faculty to adapt to new selective pressures that is consistent with observations. The model illustrates how several independent functional modules may emerge within the same protein structure, depending on the nature of past environmental fluctuations. Our model thus relates the evolutionary history of proteins to the geometry of their functional constraints, with implications for decoding and engineering protein sequences.

Kidins220/ARMS as a functional mediator of multiple receptor signalling pathways.

PubMed

Neubrand, Veronika E; Cesca, Fabrizia; Benfenati, Fabio; Schiavo, Giampietro

2012-04-15

An increasing body of evidence suggests that several membrane receptors--in addition to activating distinct signalling cascades--also engage in substantial crosstalk with each other, thereby adjusting their signalling outcome as a function of specific input information. However, little is known about the molecular mechanisms that control their coordination and integration of downstream signalling. A protein that is likely to have a role in this process is kinase-D-interacting substrate of 220 kDa [Kidins220, also known as ankyrin repeat-rich membrane spanning (ARMS), hereafter referred to as Kidins220/ARMS]. Kidins220/ARMS is a conserved membrane protein that is preferentially expressed in the nervous system and interacts with the microtubule and actin cytoskeleton. It interacts with neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, and is a common downstream target of several trophic stimuli. Kidins220/ARMS is required for neuronal differentiation and survival, and its expression levels modulate synaptic plasticity. Kidins220/ARMS knockout mice show developmental defects mainly in the nervous and cardiovascular systems, suggesting a crucial role for this protein in modulating the cross talk between different signalling pathways. In this Commentary, we summarise existing knowledge regarding the physiological functions of Kidins220/ARMS, and highlight some interesting directions for future studies on the role of this protein in health and disease.

Neurodegeneration in Alzheimer disease: role of amyloid precursor protein and presenilin 1 intracellular signaling.

PubMed

Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

2012-01-01

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

Tailoring recombinant protein quality by rational media design.

PubMed

Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé

2015-01-01

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers.

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

PubMed Central

Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

2013-01-01

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

Mitochondrial modulators in experimental Huntington's disease: reversal of mitochondrial dysfunctions and cognitive deficits.

PubMed

Mehrotra, Arpit; Kanwal, Abhinav; Banerjee, Sanjay Kumar; Sandhir, Rajat

2015-06-01

Huntington's disease (HD) is a chronic neurodegenerative condition involving impaired mitochondrial functions. The present study evaluates the therapeutic potential of combined administration of mitochondrial modulators: alpha-lipoic acid and acetyl-l-carnitine on mitochondrial dysfunctions in 3-NP-induced HD. Our results reveal 3-NP administration resulted in compromise of mitochondrial functions in terms of: (1) impaired activity of mitochondrial respiratory chain enzymes, altered cytochrome levels, reduced histochemical staining of complex-II and IV, reduced in-gel activity of complex-I to V, and reduced mRNA expression of respiratory chain complexes; (2) enhanced mitochondrial oxidative stress indicated by increased malondialdehyde, protein carbonyls, reactive oxygen species and nitrite levels, along with decreased Mn-superoxide dismutase and catalase activity; (3) mitochondrial structural changes measured by mitochondrial swelling, reduced mitochondrial membrane potential and ultra-structure changes; (4) increased cytosolic cytochrome c levels, caspase-3 and -9 activity along with altered expression of apoptotic proteins (AIF, Bim, Bad, and Bax); and (5) impaired cognitive functions assessed using Morris water maze and Y-maze. Combination of mitochondrial modulators (alpha-lipoic acid + acetyl-l-carnitine) on the other hand ameliorated 3-NP-induced mitochondrial dysfunctions, oxidative stress, histologic alterations, and behavioral deficits, suggesting their therapeutic efficacy in the management of HD. Copyright © 2015 Elsevier Inc. All rights reserved.

Analysis of Cysteine Redox Post-Translational Modifications in Cell Biology and Drug Pharmacology.

PubMed

Wani, Revati; Murray, Brion W

2017-01-01

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

Frontotemporal dementia: insights into the biological underpinnings of disease through gene co-expression network analysis.

PubMed

Ferrari, Raffaele; Forabosco, Paola; Vandrovcova, Jana; Botía, Juan A; Guelfi, Sebastian; Warren, Jason D; Momeni, Parastoo; Weale, Michael E; Ryten, Mina; Hardy, John

2016-02-24

In frontotemporal dementia (FTD) there is a critical lack in the understanding of biological and molecular mechanisms involved in disease pathogenesis. The heterogeneous genetic features associated with FTD suggest that multiple disease-mechanisms are likely to contribute to the development of this neurodegenerative condition. We here present a systems biology approach with the scope of i) shedding light on the biological processes potentially implicated in the pathogenesis of FTD and ii) identifying novel potential risk factors for FTD. We performed a gene co-expression network analysis of microarray expression data from 101 individuals without neurodegenerative diseases to explore regional-specific co-expression patterns in the frontal and temporal cortices for 12 genes (MAPT, GRN, CHMP2B, CTSC, HLA-DRA, TMEM106B, C9orf72, VCP, UBQLN2, OPTN, TARDBP and FUS) associated with FTD and we then carried out gene set enrichment and pathway analyses, and investigated known protein-protein interactors (PPIs) of FTD-genes products. Gene co-expression networks revealed that several FTD-genes (such as MAPT and GRN, CTSC and HLA-DRA, TMEM106B, and C9orf72, VCP, UBQLN2 and OPTN) were clustering in modules of relevance in the frontal and temporal cortices. Functional annotation and pathway analyses of such modules indicated enrichment for: i) DNA metabolism, i.e. transcription regulation, DNA protection and chromatin remodelling (MAPT and GRN modules); ii) immune and lysosomal processes (CTSC and HLA-DRA modules), and; iii) protein meta/catabolism (C9orf72, VCP, UBQLN2 and OPTN, and TMEM106B modules). PPI analysis supported the results of the functional annotation and pathway analyses. This work further characterizes known FTD-genes and elaborates on their biological relevance to disease: not only do we indicate likely impacted regional-specific biological processes driven by FTD-genes containing modules, but also do we suggest novel potential risk factors among the FTD-genes interactors as targets for further mechanistic characterization in hypothesis driven cell biology work.

An Autonomous BMP2 Regulatory Element in Mesenchymal Cells

PubMed Central

Kruithof, Boudewijn P.T.; Fritz, David T.; Liu, Yijun; Garsetti, Diane E.; Frank, David B.; Pregizer, Steven K.; Gaussin, Vinciane; Mortlock, Douglas P.; Rogers, Melissa B.

2014-01-01

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements. PMID:21268088

RanBPM: a potential therapeutic target for modulating diverse physiological disorders.

PubMed

Das, Soumyadip; Suresh, Bharathi; Kim, Hyongbum Henry; Ramakrishna, Suresh

2017-12-01

The Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein involved in a variety of intracellular signaling pathways that control diverse cellular functions. RanBPM interacts with proteins that are linked to various diseases, including Alzheimer's disease (AD), schizophrenia (SCZ), and cancer. In this article, we define the characteristics of the scaffolding protein RanBPM and focus on its interaction partners in diverse physiological disorders, such as neurological diseases, fertility disorders, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Biology of Non-Ribosomal Peptide Synthetases

PubMed Central

Miller, Bradley R.; Gulick, Andrew M.

2016-01-01

Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

PubMed

Creelman, Robert A.; Mullet, John E.

1997-06-01

Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

A-kinase anchoring proteins that regulate cardiac remodeling.

PubMed

Carnegie, Graeme K; Burmeister, Brian T

2011-11-01

In response to injury or stress, the adult heart undergoes maladaptive changes, collectively defined as pathological cardiac remodeling. Here, we focus on the role of A-kinase anchoring proteins (AKAPs) in 3 main areas associated with cardiac remodeling and the progression of heart failure: excitation-contraction coupling, sarcomeric regulation, and induction of pathological hypertrophy. AKAPs are a diverse family of scaffold proteins that form multiprotein complexes, integrating cAMP signaling with protein kinases, phosphatases, and other effector proteins. Many AKAPs have been characterized in the heart, where they play a critical role in modulating cardiac function.

The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis

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

Kim, Jeonghee; Chung, In Kwon, E-mail: topoviro@yonsei.ac.kr

Highlights: •Identification of U2AF65 as a novel TRF1-interacting protein. •U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. •U2AF65 interferes with the interaction between TRF1 and Fbx4. •U2AF65 represents a new route for modulating TRF1 function at telomeres. -- Abstract: The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure propermore » telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essential pre-mRNA splicing factor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.« less

Hybrid coexpression link similarity graph clustering for mining biological modules from multiple gene expression datasets.

PubMed

Salem, Saeed; Ozcaglar, Cagri

2014-01-01

Advances in genomic technologies have enabled the accumulation of vast amount of genomic data, including gene expression data for multiple species under various biological and environmental conditions. Integration of these gene expression datasets is a promising strategy to alleviate the challenges of protein functional annotation and biological module discovery based on a single gene expression data, which suffers from spurious coexpression. We propose a joint mining algorithm that constructs a weighted hybrid similarity graph whose nodes are the coexpression links. The weight of an edge between two coexpression links in this hybrid graph is a linear combination of the topological similarities and co-appearance similarities of the corresponding two coexpression links. Clustering the weighted hybrid similarity graph yields recurrent coexpression link clusters (modules). Experimental results on Human gene expression datasets show that the reported modules are functionally homogeneous as evident by their enrichment with biological process GO terms and KEGG pathways.

Comparative analysis of prophages in Streptococcus mutans genomes

PubMed Central

Fu, Tiwei; Fan, Xiangyu; Long, Quanxin; Deng, Wanyan; Song, Jinlin

2017-01-01

Prophages have been considered genetic units that have an intimate association with novel phenotypic properties of bacterial hosts, such as pathogenicity and genomic variation. Little is known about the genetic information of prophages in the genome of Streptococcus mutans, a major pathogen of human dental caries. In this study, we identified 35 prophage-like elements in S. mutans genomes and performed a comparative genomic analysis. Comparative genomic and phylogenetic analyses of prophage sequences revealed that the prophages could be classified into three main large clusters: Cluster A, Cluster B, and Cluster C. The S. mutans prophages in each cluster were compared. The genomic sequences of phismuN66-1, phismuNLML9-1, and phismu24-1 all shared similarities with the previously reported S. mutans phages M102, M102AD, and ϕAPCM01. The genomes were organized into seven major gene clusters according to the putative functions of the predicted open reading frames: packaging and structural modules, integrase, host lysis modules, DNA replication/recombination modules, transcriptional regulatory modules, other protein modules, and hypothetical protein modules. Moreover, an integrase gene was only identified in phismuNLML9-1 prophages. PMID:29158986

Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

PubMed

Saibi, Walid; Zouari, Nabil; Masmoudi, Khaled; Brini, Faiçal

2016-04-01

Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases. Copyright © 2016 Elsevier B.V. All rights reserved.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

PubMed

Wacker, Stephan Armin; Alvarado, Cristobal; von Wichert, Götz; Knippschild, Uwe; Wiedenmann, Jörg; Clauss, Karen; Nienhaus, Gerd Ulrich; Hameister, Horst; Baumann, Bernd; Borggrefe, Tilman; Knöchel, Walter; Oswald, Franz

2011-01-05

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

PubMed

Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

2014-02-01

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner*

PubMed Central

Kugler, Jamie E.; Horsch, Marion; Huang, Di; Furusawa, Takashi; Rochman, Mark; Garrett, Lillian; Becker, Lore; Bohla, Alexander; Hölter, Sabine M.; Prehn, Cornelia; Rathkolb, Birgit; Racz, Ildikó; Aguilar-Pimentel, Juan Antonio; Adler, Thure; Adamski, Jerzy; Beckers, Johannes; Busch, Dirk H.; Eickelberg, Oliver; Klopstock, Thomas; Ollert, Markus; Stöger, Tobias; Wolf, Eckhard; Wurst, Wolfgang; Yildirim, Ali Önder; Zimmer, Andreas; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; Garfinkel, Benny; Orly, Joseph; Ovcharenko, Ivan; Bustin, Michael

2013-01-01

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1tm1/tm1, Hmgn3tm1/tm1, and Hmgn5tm1/tm1 mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgntm1/tm1 lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner. PMID:23620591

The SAM domains of Anks family proteins are critically involved in modulating the degradation of EphA receptors.

PubMed

Kim, Jieun; Lee, Haeryung; Kim, Yujin; Yoo, Sooyeon; Park, Eunjeong; Park, Soochul

2010-04-01

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.

Differential role of calpain-dependent protein cleavage in intermediate and long-term operant memory in Aplysia.

PubMed

Lyons, Lisa C; Gardner, Jacob S; Lentsch, Cassidy T; Gandour, Catherine E; Krishnan, Harini C; Noakes, Eric J

2017-01-01

In addition to protein synthesis, protein degradation or protein cleavage may be necessary for intermediate (ITM) and long-term memory (LTM) to remove molecular constraints, facilitate persistent kinase activity and modulate synaptic plasticity. Calpains, a family of conserved calcium dependent cysteine proteases, modulate synaptic function through protein cleavage. We used the marine mollusk Aplysia californica to investigate the in vivo role of calpains during intermediate and long-term operant memory formation using the learning that food is inedible (LFI) paradigm. A single LFI training session, in which the animal associates a specific netted seaweed with the failure to swallow, generates short (30min), intermediate (4-6h) and long-term (24h) memory. Using the calpain inhibitors calpeptin and MDL-28170, we found that ITM requires calpain activity for induction and consolidation similar to the previously reported requirements for persistent protein kinase C activity in intermediate-term LFI memory. The induction of LTM also required calpain activity. In contrast to ITM, calpain activity was not necessary for the molecular consolidation of LTM. Surprisingly, six hours after LFI training we found that calpain activity was necessary for LTM, although this is a time at which neither persistent PKC activity nor protein synthesis is required for the maintenance of long-term LFI memory. These results demonstrate that calpains function in multiple roles in vivo during associative memory formation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cardioprotective effects of baicalein on heart failure via modulation of Ca(2+) handling proteins in vivo and in vitro.

PubMed

Zhao, Fali; Fu, Lu; Yang, Wei; Dong, Yuhui; Yang, Jing; Sun, Shoubin; Hou, Yuling

2016-01-15

Baicalein is a widely used Chinese herbal medicine extracted from Labiatae plants Scutellaria baicalensis Georgi's dry root, which has multiple pharmacological activities. However, the precise mechanism of baicalein against myocardial remodeling remains poorly understood. The aim of our study was to investigate the underlying mechanism of baicalein treatment in rats model of heart failure (HF) and rat myocardial cells (H9C2). HF model was established by abdominal aorta constriction in rats and incubation with 50μM isoproterenol for 48h in H9C2 cells. Various molecular biological experiments were performed to assess the effects of baicalein on cardiac function, myocardial remodeling, apoptosis and Ca(2+) handling proteins. In the present study, first we found that baicalein alleviated HF in vivo. Additionally, treatment with baicalein inhibited the myocardial fibrosis, restrained the expression and activity of MMP2 and MMP9, and suppressed apoptosis in heart tissue. Moreover, baicalein could inhibit the cardiac myocyte hypertrophy and apoptosis induced by isoproterenol in vitro. Finally we found that baicalein could modulate the expressions and activities of Ca(2+) handling proteins, including downregulation of phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and expression of Na(+)/Ca(2+)-exchangers (NCX1), upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and ryanodine receptor 2 (RYR2). Baicalein also restrained the decreased SERCA activity induced by aortic banding. Our studies suggested that baicalein alleviated myocardial remodeling and improved cardiac function via modulation of Ca(2+) handling proteins, which may be a potential phytochemical flavonoid for therapeutics of HF. Copyright © 2015 Elsevier Inc. All rights reserved.

Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity*

PubMed Central

Chen, Ying; Yang, Li-Na; Cheng, Li; Tu, Shun; Guo, Shu-Juan; Le, Huang-Ying; Xiong, Qian; Mo, Ran; Li, Chong-Yang; Jeong, Jun-Seop; Jiang, Lizhi; Blackshaw, Seth; Bi, Li-Jun; Zhu, Heng; Tao, Sheng-Ce; Ge, Feng

2013-01-01

Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. PMID:23824909

Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy

PubMed Central

Li, Fang; Lang, Fangfang; Zhang, Huilin; Xu, Liangdong; Wang, Yidan; Zhai, Chunxiao

2017-01-01

Apigenin, a component in daily diets, demonstrates antioxidant and anti-inflammatory properties. Here, we intended to explore the mechanism of apigenin-mediated endotoxin-induced myocardial injury and its role in the interplay among inflammation, oxidative stress, and autophagy. In our lipopolysaccharide- (LPS-) induced myocardial injury model, apigenin ameliorated cardiac injury (lactate dehydrogenase (LDH) and creatine kinase (CK)), cell death (TUNEL staining, DNA fragmentation, and PARP activity), and tissue damage (cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1)) and improved cardiac function (ejection fraction (EF) and end diastolic left ventricular inner dimension (LVID)). Apigenin also alleviated endotoxin-induced myocardial injury by modulating oxidative stress (nitrotyrosine and protein carbonyl) and inflammatory cytokines (TNF-α, IL-1β, MIP-1α, and MIP-2) along with their master regulator NFκB. Apigenin modulated redox homeostasis, and its anti-inflammatory role might be associated with its ability to control autophagy. Autophagy (determined by LAMP1, ATG5, and p62), its transcriptional regulator transcription factor EB (TFEB), and downstream target genes including vacuolar protein sorting-associated protein 11 (Vps11) and microtubule-associated proteins 1A/1B light chain 3B (Map1lc3) were modulated by apigenin. Thus, our study demonstrated that apigenin may lead to potential development of new target in sepsis treatment or other myocardial oxidative and/or inflammation-induced injuries. PMID:28828145

Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy.

PubMed

Li, Fang; Lang, Fangfang; Zhang, Huilin; Xu, Liangdong; Wang, Yidan; Zhai, Chunxiao; Hao, Enkui

2017-01-01

Apigenin, a component in daily diets, demonstrates antioxidant and anti-inflammatory properties. Here, we intended to explore the mechanism of apigenin-mediated endotoxin-induced myocardial injury and its role in the interplay among inflammation, oxidative stress, and autophagy. In our lipopolysaccharide- (LPS-) induced myocardial injury model, apigenin ameliorated cardiac injury (lactate dehydrogenase (LDH) and creatine kinase (CK)), cell death (TUNEL staining, DNA fragmentation, and PARP activity), and tissue damage (cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1)) and improved cardiac function (ejection fraction (EF) and end diastolic left ventricular inner dimension (LVID)). Apigenin also alleviated endotoxin-induced myocardial injury by modulating oxidative stress (nitrotyrosine and protein carbonyl) and inflammatory cytokines (TNF- α , IL-1 β , MIP-1 α , and MIP-2) along with their master regulator NF κ B. Apigenin modulated redox homeostasis, and its anti-inflammatory role might be associated with its ability to control autophagy. Autophagy (determined by LAMP1, ATG5, and p62), its transcriptional regulator transcription factor EB (TFEB), and downstream target genes including vacuolar protein sorting-associated protein 11 (Vps11) and microtubule-associated proteins 1A/1B light chain 3B (Map1lc3) were modulated by apigenin. Thus, our study demonstrated that apigenin may lead to potential development of new target in sepsis treatment or other myocardial oxidative and/or inflammation-induced injuries.

The Parkinson’s Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

PubMed Central

Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

2015-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

PubMed

Pin, Jean-Philippe; Acher, Francine

2002-06-01

The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these offer for drug development.

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions.

PubMed

Baxter, Robert C

2013-08-01

In addition to its important role in the regulation of somatic growth by acting as the major circulating transport protein for the insulin-like growth factors (IGFs), IGF binding protein-3 (IGFBP-3) has a variety of intracellular ligands that point to its function within major signaling pathways. The discovery of its interaction with the retinoid X receptor has led to the elucidation of roles in regulating the function of several nuclear hormone receptors including retinoic acid receptor-α, Nur77 and vitamin D receptor. Its interaction with the nuclear hormone receptor peroxisome proliferator-activated receptor-γ is believed to be involved in regulating adipocyte differentiation, which is also modulated by IGFBP-3 through an interaction with TGFβ/Smad signaling. IGFBP-3 can induce apoptosis alone or in conjunction with other agents, and in different systems can activate caspases -8 and -9. At least two unrelated proteins (LRP1 and TMEM219) have been designated as receptors for IGFBP-3, the latter with a demonstrated role in inducing caspase-8-dependent apoptosis. In contrast, IGFBP-3 also has demonstrated roles in survival-related functions, including the repair of DNA double-strand breaks through interaction with the epidermal growth factor receptor and DNA-dependent protein kinase, and the induction of autophagy through interaction with GRP78. The ability of IGFBP-3 to modulate the balance between pro-apoptotic and pro-survival sphingolipids by regulating sphingosine kinase 1 and sphingomyelinases may be integral to its role at the crossroads between cell death and survival in response to a variety of stimuli. The pleiotropic nature of IGFBP-3 activity supports the idea that IGFBP-3 itself, or pathways with which it interacts, should be investigated as targets of therapy for a variety of diseases.

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

PubMed

Wishart, Thomas M; Rooney, Timothy M; Lamont, Douglas J; Wright, Ann K; Morton, A Jennifer; Jackson, Mandy; Freeman, Marc R; Gillingwater, Thomas H

2012-01-01

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

The Functional Network of the Arabidopsis Plastoglobule Proteome Based on Quantitative Proteomics and Genome-Wide Coexpression Analysis1[C][W][OA

PubMed Central

Lundquist, Peter K.; Poliakov, Anton; Bhuiyan, Nazmul H.; Zybailov, Boris; Sun, Qi; van Wijk, Klaas J.

2012-01-01

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions. PMID:22274653

Membrane-Mediated Oligomerization of G Protein Coupled Receptors and Its Implications for GPCR Function

PubMed Central

Gahbauer, Stefan; Böckmann, Rainer A.

2016-01-01

The dimerization or even oligomerization of G protein coupled receptors (GPCRs) causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signaling complexes. Recent findings further suggest that the surrounding membrane, i.e., its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function. PMID:27826255

Modular Homogeneous Chromophore–Catalyst Assemblies

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

Mulfort, Karen L.; Utschig, Lisa M.

2016-05-17

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate thatmore » molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.« less

Cardiac myofilaments: mechanics and regulation

NASA Technical Reports Server (NTRS)

de Tombe, Pieter P.; Bers, D. M. (Principal Investigator)

2003-01-01

The mechanical properties of the cardiac myofilament are an important determinant of pump function of the heart. This report is focused on the regulation of myofilament function in cardiac muscle. Calcium ions form the trigger that induces activation of the thin filament which, in turn, allows for cross-bridge formation, ATP hydrolysis, and force development. The structure and protein-protein interactions of the cardiac sarcomere that are responsible for these processes will be reviewed. The molecular mechanism that underlies myofilament activation is incompletely understood. Recent experimental approaches have been employed to unravel the mechanism and regulation of myofilament mechanics and energetics by activator calcium and sarcomere length, as well as contractile protein phosphorylation mediated by protein kinase A. Central to these studies is the question whether such factors impact on muscle function simply by altering thin filament activation state, or whether modulation of cross-bridge cycling also plays a part in the responses of muscle to these stimuli.

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9.

PubMed

McManus, Francis P; Bourdeau, Véronique; Acevedo, Mariana; Lopes-Paciencia, Stéphane; Mignacca, Lian; Lamoliatte, Frédéric; Rojas Pino, John W; Ferbeyre, Gerardo; Thibault, Pierre

2018-05-17

Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9's ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.

HnRNP-like proteins as post-transcriptional regulators.

PubMed

Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

2014-10-01

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Probing Functional Heteromeric Chemokine Protein-Protein Interactions through Conformation-Assisted Oxime Ligation.

PubMed

Agten, Stijn M; Koenen, Rory R; Ippel, Hans; Eckardt, Veit; von Hundelshausen, Philipp; Mayo, Kevin H; Weber, Christian; Hackeng, Tilman M

2016-11-21

Protein-protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES-PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

PubMed Central

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

Phytochemicals perturb membranes and promiscuously alter protein function.

PubMed

Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

2014-08-15

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

PubMed Central

2015-01-01

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

EB-Family Proteins: Functions and Microtubule Interaction Mechanisms.

PubMed

Mustyatsa, V V; Boyakhchyan, A V; Ataullakhanov, F I; Gudimchuk, N B

2017-07-01

Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

PubMed Central

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

PubMed

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Production of functional proteins: balance of shear stress and gravity

NASA Technical Reports Server (NTRS)

Kaysen, James Howard (Inventor); Hammond, Timothy Grant (Inventor); Goodwin, Thomas John (Inventor)

2011-01-01

A method for the production of functional proteins including hormones by renal cells in a three dimensional culturing process responsive to shear stress uses a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-.alpha.-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D.sub.3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating an in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

[Not Available].

PubMed

Yanashima, Ryoji; Kitagawa, Noriyuki; Matsubara, Yoshiya; Weatheritt, Robert; Oka, Kotaro; Kikuchi, Shinichi; Tomita, Masaru; Ishizaki, Shun

2009-01-01

The scale-free and small-world network models reflect the functional units of networks. However, when we investigated the network properties of a signaling pathway using these models, no significant differences were found between the original undirected graphs and the graphs in which inactive proteins were eliminated from the gene expression data. We analyzed signaling networks by focusing on those pathways that best reflected cellular function. Therefore, our analysis of pathways started from the ligands and progressed to transcription factors and cytoskeletal proteins. We employed the Python module to assess the target network. This involved comparing the original and restricted signaling cascades as a directed graph using microarray gene expression profiles of late onset Alzheimer's disease. The most commonly used method of shortest-path analysis neglects to consider the influences of alternative pathways that can affect the activation of transcription factors or cytoskeletal proteins. We therefore introduced included k-shortest paths and k-cycles in our network analysis using the Python modules, which allowed us to attain a reasonable computational time and identify k-shortest paths. This technique reflected results found in vivo and identified pathways not found when shortest path or degree analysis was applied. Our module enabled us to comprehensively analyse the characteristics of biomolecular networks and also enabled analysis of the effects of diseases considering the feedback loop and feedforward loop control structures as an alternative path.

Normal Genetic Variation, Cognition, and Aging

PubMed Central

Greenwood, P. M.; Parasuraman, Raja

2005-01-01

This article reviews the modulation of cognitive function by normal genetic variation. Although the heritability of “g” is well established, the genes that modulate specific cognitive functions are largely unidentified. Application of the allelic association approach to individual differences in cognition has begun to reveal the effects of single nucleotide polymorphisms on specific and general cognitive functions. This article proposes a framework for relating genotype to cognitive phenotype by considering the effect of genetic variation on the protein product of specific genes within the context of the neural basis of particular cognitive domains. Specificity of effects is considered, from genes controlling part of one receptor type to genes controlling agents of neuronal repair, and evidence is reviewed of cognitive modulation by polymorphisms in dopaminergic and cholinergic receptor genes, dopaminergic enzyme genes, and neurotrophic genes. Although allelic variation in certain genes can be reliably linked to cognition—specifically to components of attention, working memory, and executive function in healthy adults—the specificity, generality, and replicability of the effects are not fully known. PMID:15006290

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

PubMed Central

Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

2013-01-01

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

SNAP-25 IN NEUROPSYCHIATRIC DISORDERS

PubMed Central

Corradini, Irene; Verderio, Claudia; Sala, Mariaelvina; Wilson, Michael C.; Matteoli, Michela

2009-01-01

SNAP-25 is plasma membrane protein which, together with syntaxin and the synaptic vesicle protein VAMP/synaptobrevin, forms the SNARE docking complex for regulated exocytosis. SNAP-25 also modulates different voltage-gated calcium channels, representing therefore a multifunctional protein that plays essential roles in neurotransmitter release at different steps. Recent genetic studies of human populations and of some mouse models implicate that alterations in SNAP-25 gene structure, expression and/or function may contribute directly to these distinct neuropsychiatric and neurological disorders. PMID:19161380

The life and miracles of kinetochores

PubMed Central

Santaguida, Stefano; Musacchio, Andrea

2009-01-01

Kinetochores are large protein assemblies built on chromosomal loci named centromeres. The main functions of kinetochores can be grouped under four modules. The first module, in the inner kinetochore, contributes a sturdy interface with centromeric chromatin. The second module, the outer kinetochore, contributes a microtubule-binding interface. The third module, the spindle assembly checkpoint, is a feedback control mechanism that monitors the state of kinetochore–microtubule attachment to control the progression of the cell cycle. The fourth module discerns correct from improper attachments, preventing the stabilization of the latter and allowing the selective stabilization of the former. In this review, we discuss how the molecular organization of the four modules allows a dynamic integration of kinetochore–microtubule attachment with the prevention of chromosome segregation errors and cell-cycle progression. PMID:19629042

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

PubMed

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

Lipid Interaction Sites on Channels, Transporters and Receptors: Recent Insights from Molecular Dynamics Simulations

PubMed Central

Hedger, George; Sansom, Mark S. P.

2017-01-01

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterisation of these sites is of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. PMID:26946244

When galectins recognize glycans: from biochemistry to physiology and back again.

PubMed

Di Lella, Santiago; Sundblad, Victoria; Cerliani, Juan P; Guardia, Carlos M; Estrin, Dario A; Vasta, Gerardo R; Rabinovich, Gabriel A

2011-09-20

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings. © 2011 American Chemical Society

Tetramer formation of tumor suppressor protein p53: Structure, function, and applications.

PubMed

Kamada, Rui; Toguchi, Yu; Nomura, Takao; Imagawa, Toshiaki; Sakaguchi, Kazuyasu

2016-11-04

Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal, and reprogramming of differentiated cells into stem cells, immune system, and metastasis. More than 50% of human tumors have TP53 gene mutations, and most of them are missense mutations that presumably reduce tumor suppressor activity of p53. This review focuses on the role of the tetramerization (oligomerization), which is modulated by the protein concentration of p53, posttranslational modifications, and/or interactions with its binding proteins, in regulating the tumor suppressor function of p53. Functional control of p53 by stabilizing or inhibiting oligomer formation and its bio-applications are also discussed. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 598-612, 2016. © 2015 Wiley Periodicals, Inc.

Posttranslational modification of autophagy-related proteins in macroautophagy

PubMed Central

Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J.; Tang, Daolin

2014-01-01

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases. PMID:25484070

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

PubMed

Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

2014-01-01

Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters

PubMed Central

Zhang, Minhua; Bao, Zhihao; Zhao, Qin; Guo, Hui; Xu, Ke; Wang, Chengcheng

2014-01-01

Energy-coupling factor (ECF) transporters are a unique group of ATP-binding cassette (ABC) transporters responsible for micronutrient uptake from the environment. Each ECF transporter is composed of an S component (or EcfS protein) and T/A/A′ components (or EcfT/A/A′ proteins; ECF module). Among the group II ECF transporters, several EcfS proteins share one ECF module; however, the underlying mechanism remains unknown. Here we report the structure of a group II ECF transporter–pantothenate transporter from Lactobacillus brevis (LbECF-PanT), which shares the ECF module with the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT). Structural and mutational analyses revealed the residues constituting the pantothenate-binding pocket. We found that although the three EcfS proteins PanT, FolT, and HmpT are dissimilar in sequence, they share a common surface area composed of the transmembrane helices 1/2/6 (SM1/2/6) to interact with the coupling helices 2/3 (CH2/3) of the same EcfT. CH2 interacts mainly with SM1 via hydrophobic interactions, which may modulate the sliding movement of EcfS. CH3 binds to a hydrophobic surface groove formed by SM1, SM2, and SM6, which may transmit the conformational changes from EcfA/A′ to EcfS. We also found that the residues at the intermolecular surfaces in LbECF-PanT are essential for transporter activity, and that these residues may mediate intermolecular conformational transmission and/or affect transporter complex stability. In addition, we found that the structure of EcfT is conformationally dynamic, which supports its function as a scaffold to mediate the interaction of the ECF module with various EcfS proteins to form different transporter complexes. PMID:25512487

Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

PubMed

Butler, David; Hwang, Jeannie; Estick, Candice; Nishiyama, Akiko; Kumar, Saranya Santhosh; Baveghems, Clive; Young-Oxendine, Hollie B; Wisniewski, Meagan L; Charalambides, Ana; Bahr, Ben A

2011-01-01

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.

Genomic Heterogeneity of Osteosarcoma - Shift from Single Candidates to Functional Modules

PubMed Central

Maugg, Doris; Eckstein, Gertrud; Baumhoer, Daniel; Nathrath, Michaela; Korsching, Eberhard

2015-01-01

Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and can here be exemplified for OS. PMID:25848766

Automating tasks in protein structure determination with the clipper python module.

PubMed

McNicholas, Stuart; Croll, Tristan; Burnley, Tom; Palmer, Colin M; Hoh, Soon Wen; Jenkins, Huw T; Dodson, Eleanor; Cowtan, Kevin; Agirre, Jon

2018-01-01

Scripting programming languages provide the fastest means of prototyping complex functionality. Those with a syntax and grammar resembling human language also greatly enhance the maintainability of the produced source code. Furthermore, the combination of a powerful, machine-independent scripting language with binary libraries tailored for each computer architecture allows programs to break free from the tight boundaries of efficiency traditionally associated with scripts. In the present work, we describe how an efficient C++ crystallographic library such as Clipper can be wrapped, adapted and generalized for use in both crystallographic and electron cryo-microscopy applications, scripted with the Python language. We shall also place an emphasis on best practices in automation, illustrating how this can be achieved with this new Python module. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Isolation and characterizations of oxalate-binding proteins in the kidney

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

Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta

Highlights: Black-Right-Pointing-Pointer The first large-scale characterizations of oxalate-binding kidney proteins. Black-Right-Pointing-Pointer The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. Black-Right-Pointing-Pointer 38 forms of 26 unique oxalate-binding kidney proteins were identified. Black-Right-Pointing-Pointer 25/26 (96%) of identified proteins had 'L-x(3,5)-R-x(2)-[AGILPV]' domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) tomore » resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed 'L-x(3,5)-R-x(2)-[AGILPV]' as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.« less

Measuring ECS Interaction with Biomembranes.

PubMed

Angelucci, Clotilde B; Sabatucci, Annalaura; Dainese, Enrico

2016-01-01

Understanding the correct interaction among the different components of the endocannabinoid system (ECS) is fundamental for a proper assessment of the function of endocannabinoids (eCBs) as signaling molecules. The knowledge of how membrane environment is able to modulate intracellular trafficking of eCBs and their interacting proteins holds a huge potential in unraveling new mechanisms of ECS modulation.Here, fluorescence resonance energy transfer (FRET) technique is applied to measure the binding affinity of ECS proteins to model membranes (i.e., large unilamellar vesicles, LUVs). In particular, we describe in details the paradigmatic example of the interaction of recombinant rat FAAH-ΔTM with LUVs constituted by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

Membrane trafficking pathways and their roles in plant-microbe interactions.

PubMed

Inada, Noriko; Ueda, Takashi

2014-04-01

Membrane trafficking functions in the delivery of proteins that are newly synthesized in the endoplasmic reticulum (ER) to their final destinations, such as the plasma membrane (PM) and the vacuole, and in the internalization of extracellular components or PM-associated proteins for recycling or degradative regulation. These trafficking pathways play pivotal roles in the rapid responses to environmental stimuli such as challenges by microorganisms. In this review, we provide an overview of the current knowledge of plant membrane trafficking and its roles in plant-microbe interactions. Although there is little information regarding the mechanism of pathogenic modulation of plant membrane trafficking thus far, recent research has identified many membrane trafficking factors as possible targets of microbial modulation.

Mechanisms, biology and inhibitors of deubiquitinating enzymes.

PubMed

Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L

2007-11-01

The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

Melatonin modulates rat myotube-acetylcholine receptors by inhibiting calmodulin.

PubMed

de Almeida-Paula, Lidiana Duarte; Costa-Lotufo, Leticia V; Silva Ferreira, Zulma; Monteiro, Amanda Elisa G; Isoldi, Mauro Cesar; Godinho, Rosely O; Markus, Regina P

2005-11-21

Melatonin, the pineal gland hormone, modulates alpha-bungarotoxin sensitive nicotinic acetylcholine receptors in sympathetic nerve terminals, cerebellum and chick retina imposing a diurnal variation in functional responses [Markus, R.P., Zago, W.M., Carneiro, R.C., 1996. Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 279, 18-22; Markus, R.P., Santos, J.M., Zago, W., Reno, L.A., 2003. Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [3HI] glutamate release in rat cerebellum slices. J. Pharmacol. Exp. Ther. 305, 525-530; Sampaio, L.F.S., Hamassaki-Britto, D.E., Markus, R.P., 2005. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells. Braz. J. Med. Biol. Res. 38, 603-613]. Here we show that in rat myotubes forskolin and melatonin reduced the number of nicotinic acetylcholine receptors expressed in plasma membrane. In addition, these cells expressed melatonin MT1 receptors, which are known to be coupled to G(i)-protein. However, the pharmacological profile of melatonin analogs regarding the reduction in cyclic AMP accumulation and number of nicotinic acetylcholine receptors did not point to a mechanism mediated by activation of G(i)-protein coupled receptors. On the other hand, calmidazolium, a classical inhibitor of calmodulin, reduced in a similar manner both effects. Considering that one isoform of adenylyl cyclase present in rat myotubes is regulated by Ca2+/calmodulin, we propose that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

PubMed

Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

2018-05-15

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

AES/GRG5: more than just a dominant-negative TLE/GRG family member.

PubMed

Beagle, Brandon; Johnson, Gail V W

2010-11-01

The human Transducin-like Enhancer of Split (TLE) and mouse homologue, Groucho gene-related protein (GRG), represent a family of conserved non-DNA binding transcriptional modulatory proteins divided into two subgroups based upon size. The long TLE/GRGs consist of four pentadomain proteins that are dedicated co-repressors for multiple transcription factors (TF). The second TLE/GRG subgroup is composed of the Amino-terminal Enhancer of Split (AES) in humans and its mouse homolog GRG5 (AES/GRG5). In contrast to the dedicated co-repressor function of long TLE/GRGs, AES/GRG5 can both positively or negatively modulate various TF as well as non-TF proteins in a long TLE/GRG-dependent or -independent manner. Therefore, AES/GRG5 is a functionally dynamic protein that is not exclusively defined by its role as a long TLE/GRG antagonist. AES/GRG5 may function in various developmental and pathological processes but the functional characteristics of endogenous AES/GRG5 in a physiologically relevant context remains to be determined. © 2010 Wiley-Liss, Inc.

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.

Methods in Enzymology: “Flexible backbone sampling methods to model and design protein alternative conformations”

PubMed Central

Ollikainen, Noah; Smith, Colin A.; Fraser, James S.; Kortemme, Tanja

2013-01-01

Sampling alternative conformations is key to understanding how proteins work and engineering them for new functions. However, accurately characterizing and modeling protein conformational ensembles remains experimentally and computationally challenging. These challenges must be met before protein conformational heterogeneity can be exploited in protein engineering and design. Here, as a stepping stone, we describe methods to detect alternative conformations in proteins and strategies to model these near-native conformational changes based on backrub-type Monte Carlo moves in Rosetta. We illustrate how Rosetta simulations that apply backrub moves improve modeling of point mutant side chain conformations, native side chain conformational heterogeneity, functional conformational changes, tolerated sequence space, protein interaction specificity, and amino acid co-variation across protein-protein interfaces. We include relevant Rosetta command lines and RosettaScripts to encourage the application of these types of simulations to other systems. Our work highlights that critical scoring and sampling improvements will be necessary to approximate conformational landscapes. Challenges for the future development of these methods include modeling conformational changes that propagate away from designed mutation sites and modulating backbone flexibility to predictively design functionally important conformational heterogeneity. PMID:23422426

Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

PubMed

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-10-02

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of interaction of mutant TP53 and wild type BRCA1 by alkaloids: a computational approach towards targeting protein-protein interaction as a futuristic therapeutic intervention strategy for breast cancer impediment.

PubMed

Tiwari, Sameeksha; Awasthi, Manika; Singh, Swati; Pandey, Veda P; Dwivedi, Upendra N

2017-10-23

Protein-protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein-protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein-protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski's rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.

Interaction of small G protein signaling modulator 3 with connexin 43 contributes to myocardial infarction in rat hearts.

PubMed

Lee, Chang Youn; Choi, Jung-Won; Shin, Sunhye; Lee, Jiyun; Seo, Hyang-Hee; Lim, Soyeon; Lee, Seahyoung; Joo, Hyun-Chul; Kim, Sang Woo; Hwang, Ki-Chul

2017-09-16

Connexin 43 (Cx43), a ubiquitous connexin expressed in the heart and skin, is associated with a variety of hereditary conditions. Therefore, the characterization of Cx43-interacting proteins and their dynamics is important to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication but also to identify novel and unanticipated biological functions of Cx43. In the present study, we observed potential targets of Cx43 to determine new molecular functions in cardio-protection. MALDI-TOF mass spectrometry analysis of Cx43 co-immunoprecipitated proteins showed that Cx43 interacts with several proteins related to metabolism. In GeneMANIA network analysis, SGSM3, which has not been previously associated with Cx43, was highly correlated with Cx43 in heart functions, and high levels of SGSM3 appeared to induce the turnover of Cx43 through lysosomal degradation in myocardial infarcted rat hearts. Moreover, we confirmed that lysosomal degradation of Cx43 is dependent upon the interaction between SGSM3 and Cx43 in H9c2 cardiomyocytes. The functional importance of the interaction between SGSM3 and Cx43 was confirmed by results showing that Cx43 expression was enhanced by SGSM3 siRNA knockdown in H9c2 cells. In summary, the results of this study elucidate the molecular mechanisms in which Cx43 with SGSM3 is degraded in myocardial infarcted rat hearts, which may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

PubMed Central

Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K

2014-01-01

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. PMID:25033834

LenVarDB: database of length-variant protein domains.

PubMed

Mutt, Eshita; Mathew, Oommen K; Sowdhamini, Ramanathan

2014-01-01

Protein domains are functionally and structurally independent modules, which add to the functional variety of proteins. This array of functional diversity has been enabled by evolutionary changes, such as amino acid substitutions or insertions or deletions, occurring in these protein domains. Length variations (indels) can introduce changes at structural, functional and interaction levels. LenVarDB (freely available at http://caps.ncbs.res.in/lenvardb/) traces these length variations, starting from structure-based sequence alignments in our Protein Alignments organized as Structural Superfamilies (PASS2) database, across 731 structural classification of proteins (SCOP)-based protein domain superfamilies connected to 2 730 625 sequence homologues. Alignment of sequence homologues corresponding to a structural domain is available, starting from a structure-based sequence alignment of the superfamily. Orientation of the length-variant (indel) regions in protein domains can be visualized by mapping them on the structure and on the alignment. Knowledge about location of length variations within protein domains and their visual representation will be useful in predicting changes within structurally or functionally relevant sites, which may ultimately regulate protein function. Non-technical summary: Evolutionary changes bring about natural changes to proteins that may be found in many organisms. Such changes could be reflected as amino acid substitutions or insertions-deletions (indels) in protein sequences. LenVarDB is a database that provides an early overview of observed length variations that were set among 731 protein families and after examining >2 million sequences. Indels are followed up to observe if they are close to the active site such that they can affect the activity of proteins. Inclusion of such information can aid the design of bioengineering experiments.

Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

PubMed

Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

2011-04-01

Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles.

α7nAchR/NMDAR coupling affects NMDAR function and object recognition.

PubMed

Li, Shupeng; Nai, Qiang; Lipina, Tatiana V; Roder, John C; Liu, Fang

2013-12-20

The α7 nicotinic acetylcholine receptor (nAchR) and NMDA glutamate receptor (NMDAR) are both ligand-gated ion channels permeable to Ca2+ and Na+. Previous studies have demonstrated functional modulation of NMDARs by nAchRs, although the molecular mechanism remains largely unknown. We have previously reported that α7nAchR forms a protein complex with the NMDAR through a protein-protein interaction. We also developed an interfering peptide that is able to disrupt the α7nAchR-NMDAR complex and blocks cue-induced reinstatement of nicotine-seeking in rat models of relapse. In the present study, we investigated whether the α7nAchR-NMDAR interaction is responsible for the functional modulation of NMDAR by α7nAchR using both electrophysiological and behavioral tests. We have found that activation of α7nAchR upregulates NMDAR-mediated whole cell currents and LTP of mEPSC in cultured hippocampal neurons, which can be abolished by the interfering peptide that disrupts the α7nAchR-NMDAR interaction. Moreover, administration of the interfering peptide in mice impairs novel object recognition but not Morris water maze performance. Our results suggest that α7nAchR/NMDAR coupling may selectively affect some aspects of learning and memory.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

PubMed Central

Yang, Di; Fletcher, Sally C.; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J.

2017-01-01

Abstract Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. PMID:28973444

Scale-space measures for graph topology link protein network architecture to function.

PubMed

Hulsman, Marc; Dimitrakopoulos, Christos; de Ridder, Jeroen

2014-06-15

The network architecture of physical protein interactions is an important determinant for the molecular functions that are carried out within each cell. To study this relation, the network architecture can be characterized by graph topological characteristics such as shortest paths and network hubs. These characteristics have an important shortcoming: they do not take into account that interactions occur across different scales. This is important because some cellular functions may involve a single direct protein interaction (small scale), whereas others require more and/or indirect interactions, such as protein complexes (medium scale) and interactions between large modules of proteins (large scale). In this work, we derive generalized scale-aware versions of known graph topological measures based on diffusion kernels. We apply these to characterize the topology of networks across all scales simultaneously, generating a so-called graph topological scale-space. The comprehensive physical interaction network in yeast is used to show that scale-space based measures consistently give superior performance when distinguishing protein functional categories and three major types of functional interactions-genetic interaction, co-expression and perturbation interactions. Moreover, we demonstrate that graph topological scale spaces capture biologically meaningful features that provide new insights into the link between function and protein network architecture. Matlab(TM) code to calculate the scale-aware topological measures (STMs) is available at http://bioinformatics.tudelft.nl/TSSA © The Author 2014. Published by Oxford University Press.

Gene Fusion: A Genome Wide Survey

NASA Technical Reports Server (NTRS)

Liang, Ping; Riley, Monica

2001-01-01

As a well known fact, organisms form larger and complex multimodular (composite or chimeric) and mostly multi-functional proteins through gene fusion of two or more individual genes which have independent evolution histories and functions. We call each of these components a module. The existence of multimodular proteins may improves the efficiency in gene regulation and in cellular functions, and thus may give the host organism advantages in adaptation to environments. Analysis of all gene fusions in present-day organisms should allow us to examine the patterns of gene fusion in context with cellular functions, to trace back the evolution processes from the ancient smaller and uni-functional proteins to the present-day larger and complex multi-functional proteins, and to estimate the minimal number of ancestor proteins that existed in the last common ancestor for all life on earth. Although many multimodular proteins have been experimentally known, identification of gene fusion events systematically at genome scale had not been possible until recently when large number of completed genome sequences have been becoming available. In addition, technical difficulties for such analysis also exist due to the complexity of this biological and evolutionary process. We report from this study a new strategy to computationally identify multimodular proteins using completed genome sequences and the results surveyed from 22 organisms with the data from over 40 organisms to be presented during the meeting. Additional information is contained in the original extended abstract.

Polypyrimidine tract-binding protein influences negative strand RNA synthesis of dengue virus.

PubMed

Jiang, Linbin; Yao, Huiling; Duan, Xiaoqun; Lu, Xi; Liu, Yongming

2009-07-24

Flavivirus non-structural protein 4A (NS4A) induces membrane rearrangements to form viral replication complex and functions as interferon antagonist. However, other non-structural roles of NS4A protein in relation to virus life-cycle are poorly defined. This study elucidated if dengue virus (DENV) NS4A protein interacts with host proteins and contributes to viral pathogenesis by screening human liver cDNA yeast-two-hybrid library. Our study identified polypyrimidine tract-binding protein (PTB) as a novel interacting partner of DENV NS4A protein. We reported for the first time that PTB influenced DENV production. Gene-silencing studies showed that PTB did not have an effect on DENV entry and DENV RNA translation. Further functional studies revealed that PTB influenced DENV production by modulating negative strand RNA synthesis. This is the first study that enlightens the interaction of DENV NS4A protein with PTB, in addition to demonstrating the novel role of PTB in relation to mosquito-borne flavivirus life-cycle.

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins.

PubMed

Norman, Michael; Rivers, Caroline; Lee, Youn-Bok; Idris, Jalilah; Uney, James

2016-12-01

RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein-protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins. © 2016 The Author(s).

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lynham, Steven; Garcia-Manyes, Sergi

2016-08-01

The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.

Modulating the protein content of complex proteomes using acetonitrile.

PubMed

Prates, João; Martins, Gonçalo; López-Fernández, Hugo; Lodeiro, Carlos; Capelo, J L; Santos, Hugo M

2018-05-15

In this work we present acetonitrile as a tool to modulate the dynamic range of the proteome of complex samples. Different concentrations of acetonitrile ranging from 15% v/v to 65% v/v were used to modulate the protein content of serum samples from healthy people and patients with lymphoma and myeloma. We show that the proteome above 70 kDa is pelleted as a function of the concentration of acetonitrile and that profiling with PCA or Clustering is only possible using the supernatants obtained for concentrations of acetonitrile higher than 45% v/v or the pellets for concentrations of acetonitrile of 35% and 45%. The differentiation and classification of the three groups of sera samples (healthy, lymphoma and myeloma) were possible using acetonitrile at 55% v/v concentration. This work opens new avenues for the application of acetonitrile as a cost-effective tool in proteomics applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescence-based assay probing regulator of G protein signaling partner proteins.

PubMed

Huang, Po-Shiun; Yeh, Hsin-Sung; Yi, Hsiu-Ping; Lin, Chain-Jia; Yang, Chii-Shen

2012-04-01

The regulator of G protein signaling (RGS) proteins are one of the essential modulators for the G protein system. Besides regulating G protein signaling by accelerating the GTPase activity of Gα subunits, RGS proteins are implicated in exerting other functions; they are also known to be involved in several diseases. Moreover, the existence of a single RGS protein in plants and its seven-transmembrane domain found in 2003 triggered efforts to unveil detailed structural and functional information of RGS proteins. We present a method for real-time examination of the protein-protein interactions between RGS and Gα subunits. AtRGS1 from plants and RGS4 from mammals were site-directedly labeled with the fluorescent probe Lucifer yellow on engineered cysteine residues and used to interact with different Gα subunits. The physical interactions can be revealed by monitoring the real-time fluorescence changes (8.6% fluorescence increase in mammals and 27.6% in plants); their correlations to functional exertion were shown with a GTPase accelerating activity assay and further confirmed by measurement of K(d). We validate the effectiveness of this method and suggest its application to the exploration of more RGS signaling partner proteins in physiological and pathological studies. Copyright © 2012 Elsevier Inc. All rights reserved.

The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.

PubMed

Child, Emma S; Mann, David J

2006-06-01

p21 was originally described as functioning as a cell cycle regulator via inhibition of both cyclin-dependent kinases and processive DNA replication. Nowadays it is recognized to play other fundamental roles including transcriptional regulation and the modulation of apoptosis. Each of these functions of p21 is achieved through direct p21/protein interactions and the subcellular localization of p21 plays an important part in dictating the binding partners to which p21 is exposed. Over recent years, a number of phosphorylation sites in p21 have been identified, these being targeted by several important intracellular signalling protein kinases. Here we review the state of our knowledge of p21 phosphorylation with respect to the kinases involved and the molecular biological effects of each phosphorylation event.

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.

Cavin Family: New Players in the Biology of Caveolae.

PubMed

Nassar, Zeyad D; Parat, Marie-Odile

2015-01-01

Caveolae are specialized small plasma-membrane invaginations that play crucial cellular functions. Two essential protein families are required for caveola formation: membrane caveolin proteins and cytoplasmic cavin proteins. Each family includes members with specific tissue distribution, and their expression is altered under physiological and pathological conditions, implying highly specialized functions. Cavins not only stabilize caveolae, but modulate their morphology and functions as well. Before association with the plasma membrane, cavins form homo- and hetero-oligomers with strikingly strict stoichiometry in the cytosol. At the plasma membrane, they provide an outer peripheral cytosolic layer, necessary for caveola stability. Interestingly, upon stimulation, cavins can be released from caveolae into the cytoplasm in distinct subcomplexes, providing a rapid dynamic link between caveolae and cellular organelles including the nucleus. In this review, we detail the biology of cavins, their structural and functional roles, and their implication in pathophysiology. Copyright © 2015 Elsevier Inc. All rights reserved.

Multifarious Functions of the Fragile X Mental Retardation Protein.

PubMed

Davis, Jenna K; Broadie, Kendal

2017-10-01

Fragile X syndrome (FXS), a heritable intellectual and autism spectrum disorder (ASD), results from the loss of Fragile X mental retardation protein (FMRP). This neurodevelopmental disease state exhibits neural circuit hyperconnectivity and hyperexcitability. Canonically, FMRP functions as an mRNA-binding translation suppressor, but recent findings have enormously expanded its proposed roles. Although connections between burgeoning FMRP functions remain unknown, recent advances have extended understanding of its involvement in RNA, channel, and protein binding that modulate calcium signaling, activity-dependent critical period development, and the excitation-inhibition (E/I) neural circuitry balance. In this review, we contextualize 3 years of FXS model research. Future directions extrapolated from recent advances focus on discovering links between FMRP roles to determine whether FMRP has a multitude of unrelated functions or whether combinatorial mechanisms can explain its multifaceted existence. Copyright © 2017 Elsevier Ltd. All rights reserved.

The structure of mouse cytomegalovirus m04 protein obtained from sparse NMR data reveals a conserved fold of the m02-m06 viral immune modulator family.

PubMed

Sgourakis, Nikolaos G; Natarajan, Kannan; Ying, Jinfa; Vogeli, Beat; Boyd, Lisa F; Margulies, David H; Bax, Ad

2014-09-02

Immunoevasins are key proteins used by viruses to subvert host immune responses. Determining their high-resolution structures is key to understanding virus-host interactions toward the design of vaccines and other antiviral therapies. Mouse cytomegalovirus encodes a unique set of immunoevasins, the m02-m06 family, that modulates major histocompatibility complex class I (MHC-I) antigen presentation to CD8+ T cells and natural killer cells. Notwithstanding the large number of genetic and functional studies, the structural biology of immunoevasins remains incompletely understood, largely because of crystallization bottlenecks. Here we implement a technology using sparse nuclear magnetic resonance data and integrative Rosetta modeling to determine the structure of the m04/gp34 immunoevasin extracellular domain. The structure reveals a β fold that is representative of the m02-m06 family of viral proteins, several of which are known to bind MHC-I molecules and interfere with antigen presentation, suggesting its role as a diversified immune regulation module. Copyright © 2014 Elsevier Ltd. All rights reserved.

β2-adrenoceptor-induced modulation of transglutaminase 2 transamidase activity in cardiomyoblasts.

PubMed

Vyas, Falguni S; Nelson, Carl P; Freeman, Fiona; Boocock, David J; Hargreaves, Alan J; Dickenson, John M

2017-10-15

Tissue transglutaminase 2 (TG2) is modulated by protein kinase A (PKA) mediated phosphorylation: however, the precise mechanism(s) of its modulation by G-protein coupled receptors coupled to PKA activation are not fully understood. In the current study we investigated the potential regulation of TG2 activity by the β 2 -adrenoceptor in rat H9c2 cardiomyoblasts. Transglutaminase transamidation activity was assessed using amine-incorporating and protein cross-linking assays. TG2 phosphorylation was determined via immunoprecipitation and Western blotting. The long acting β 2 -adrenoceptor agonist formoterol induced time- and concentration-dependent increases in TG2 transamidation. Increases in TG2 activity were reduced by the TG2 inhibitors Z-DON (Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 ((1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride). Responses to formoterol were blocked by pharmacological inhibition of PKA, extracellular signal-regulated kinase 1 and 2 (ERK1/2), or phosphatidylinositol 3-kinase (PI-3K) signalling. Furthermore, the removal of extracellular Ca 2+ also attenuated formoterol-induced TG2 activation. Fluorescence microscopy demonstrated TG2-induced biotin-X-cadaverine incorporation into proteins. Formoterol increased the levels of TG2-associated phosphoserine and phosphothreonine, which were blocked by inhibition of PKA, ERK1/2 or PI-3K signalling. Subsequent proteomic analysis identified known (e.g. lactate dehydrogenase A chain) and novel (e.g. Protein S100-A6) protein substrates for TG2. Taken together, the data obtained suggest that β 2 -adrenoceptor-induced modulation of TG2 represents a novel paradigm in β 2 -adrenoceptor cell signalling, expanding the repertoire of cellular functions responsive to catecholamine stimulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease.

PubMed

Zhou, Xiao; Li, Gang; Kaplan, Anna; Gaschler, Michael M; Zhang, Xiaoyan; Hou, Zhipeng; Jiang, Mali; Zott, Roseann; Cremers, Serge; Stockwell, Brent R; Duan, Wenzhen

2018-05-01

Huntington's disease (HD) is caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene encoding an elongated polyglutamine tract within the N-terminal of the huntingtin protein (Htt) and leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress by mutant Htt (mHtt) results in cellular dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a chaperone protein located in the ER. Our previous studies demonstrated that mHtt caused PDI to accumulate at mitochondria-associated ER membranes and triggered cell death, and that modulating PDI activity using small molecules protected cells again mHtt toxicity in cell and brain slice models of HD. In this study, we demonstrated that PDI is upregulated in the HD human brain, in cell and mouse models. Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171-82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.

Interplay between grain structure and protein adsorption on functional response of osteoblasts: ultrafine-grained versus coarse-grained substrates.

PubMed

Misra, R D K; Nune, C; Pesacreta, T C; Somani, M C; Karjalainen, L P

2013-01-01

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions. Copyright © 2012 Wiley Periodicals, Inc.

Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

PubMed Central

Ren, Jun; Zhou, Wei; Wang, Jianxin

2014-01-01

Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

PubMed

Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu

2016-01-01

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.