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Sample records for interacting sr proteins

  1. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  2. Interaction of Transportin-SR2 with Ras-related Nuclear Protein (Ran) GTPase*

    PubMed Central

    Taltynov, Oliver; Demeulemeester, Jonas; Christ, Frauke; De Houwer, Stéphanie; Tsirkone, Vicky G.; Gerard, Melanie; Weeks, Stephen D.; Strelkov, Sergei V.; Debyser, Zeger

    2013-01-01

    The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are capable of infecting non-dividing cells and, therefore, need to be imported into the nucleus before integration into the host cell chromatin. Transportin-SR2 (TRN-SR2, Transportin-3, TNPO3) is a cellular karyopherin implicated in nuclear import of HIV-1. A model in which TRN-SR2 imports the viral preintegration complex into the nucleus is supported by direct interaction between TRN-SR2 and HIV-1 integrase (IN). Residues in the C-terminal domain of HIV-1 IN that mediate binding to TRN-SR2 were recently delineated. As for most nuclear import cargoes, the driving force behind HIV-1 preintegration complex import is likely a gradient of the GDP- and GTP-bound forms of Ran, a small GTPase. In this study we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. By size exclusion chromatography we demonstrate stable complex formation of TRN-SR2 and RanGTP in solution. Consistent with the behavior of normal nuclear import cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. Although in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2-RanGTP complex was significantly more compact. Further analysis supported a model wherein one monomer of TRN-SR2 is bound to one monomer of RanGTP. Finally, we present a homology model of the TRN-SR2-RanGTP complex that is in excellent agreement with the experimental small angle x-ray scattering data. PMID:23878195

  3. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    SciTech Connect

    A.S.N. Reddy

    2008-11-25

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  4. MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

    PubMed

    Caetano, Fabiana A; Dirk, Brennan S; Tam, Joshua H K; Cavanagh, P Craig; Goiko, Maria; Ferguson, Stephen S G; Pasternak, Stephen H; Dikeakos, Jimmy D; de Bruyn, John R; Heit, Bryan

    2015-12-01

    Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell. PMID:26657340

  5. MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures

    PubMed Central

    Caetano, Fabiana A.; Dirk, Brennan S.; Tam, Joshua H. K.; Cavanagh, P. Craig; Goiko, Maria; Ferguson, Stephen S. G.; Pasternak, Stephen H.; Dikeakos, Jimmy D.; de Bruyn, John R.; Heit, Bryan

    2015-01-01

    Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell. PMID:26657340

  6. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  7. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  8. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  9. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6.

    PubMed

    Soell, M; Holveck, F; Schöller, M; Wachsmann, R D; Klein, J P

    1994-05-01

    To examine the possible implication of protein SR, an I/II-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-alpha release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-alpha. PMID:8168943

  10. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6.

    PubMed Central

    Soell, M; Holveck, F; Schöller, M; Wachsmann, R D; Klein, J P

    1994-01-01

    To examine the possible implication of protein SR, an I/II-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-alpha release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-alpha. Images PMID:8168943

  11. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  12. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  13. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  14. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  15. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  16. Carotenoid-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Britton, George; Helliwell, John R.

    Chapter 5 shows that the aggregation of carotenoid molecules can have a profound effect on their properties and hence their functioning in biological systems. Another important influence is the interaction between carotenoids and other molecules. The way that interactions of carotenoids with lipid bilayers influence the structure and properties of membranes and membrane-asociated processes is discussed in Chapter 10, and the aggregation of carotenoid molecules within the bilayers in Chapter 5. Of particular importance, though, are interactions between carotenoids and proteins. These allow the hydrophobic carotenoids to be transported, to exist, and to function in an aqueous environment. In some cases they may modify strongly the light-absorption properties and hence the colour and photochemistry of the carotenoids.

  17. Facet-Specific Assembly of Proteins on SrTiO3 Polyhedral Nanocrystals

    PubMed Central

    Dong, Lingqing; Luo, Qi; Cheng, Kui; Shi, Hui; Wang, Qi; Weng, Wenjian; Han, Wei-Qiang

    2014-01-01

    Precisely controlling the protein-nanomaterial interactions at selective sites is crucial in engineering biomolecule composite architectures with tailored nanostructures and functions for a variety of biomedical applications. This strategy, however, is only beginning to be explored. Here, we demonstrate the facet-specific assembly of proteins, such as albumin, immunoglobulin and protamine, on {100} facets of SrTiO3 polyhedral nanocrystals, while none on {110} facets. Molecular dynamics simulations indicate the immobile surface hydration layer might play a barrier role to effectively prevent proteins adsorption on specific {110} facets. This work thus provides new insights into the fundamentally understanding of protein-nanomaterial interactions, and open a novel, general and facile route to control the selective adsorption of various proteins on various nanocrystals. PMID:24866740

  18. Nanobiotechnology: protein-nanomaterial interactions.

    PubMed

    Kane, Ravi S; Stroock, Abraham D

    2007-01-01

    We review recent research that involves the interaction of nanomaterials such as nanoparticles, nanowires, and carbon nanotubes with proteins. We begin with a focus on the fundamentals of the structure and function of proteins on nanomaterials. We then review work in three areas that exploit these interactions: (1) sensing, (2) assembly of nanomaterials by proteins and proteins by nanomaterials, and (3) interactions with cells. We conclude with the identification of challenges and opportunities for the future. PMID:17335286

  19. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  20. Imaging Protein-protein Interactions in vivo

    PubMed Central

    Seegar, Tom; Barton, William

    2010-01-01

    Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface. PMID:20972411

  1. Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation.

    PubMed Central

    Tang, Zhaohua; Käufer, Norbert F; Lin, Ren-Jang

    2002-01-01

    The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast. PMID:12186627

  2. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

    PubMed

    Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

    2016-02-01

    Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

  3. The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

    PubMed

    Carvalho, Raquel F; Szakonyi, Dóra; Simpson, Craig G; Barbosa, Inês C R; Brown, John W S; Baena-González, Elena; Duque, Paula

    2016-08-01

    The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars. PMID:27436712

  4. SR proteins are required for nematode trans-splicing in vitro.

    PubMed Central

    Sanford, J R; Bruzik, J P

    1999-01-01

    SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts. PMID:10411135

  5. PINT: Protein-protein Interactions Thermodynamic Database.

    PubMed

    Kumar, M D Shaji; Gromiha, M Michael

    2006-01-01

    The first release of Protein-protein Interactions Thermodynamic Database (PINT) contains >1500 data of several thermodynamic parameters along with sequence and structural information, experimental conditions and literature information. Each entry contains numerical data for the free energy change, dissociation constant, association constant, enthalpy change, heat capacity change and so on of the interacting proteins upon binding, which are important for understanding the mechanism of protein-protein interactions. PINT also includes the name and source of the proteins involved in binding, their Protein Information Resource, SWISS-PROT and Protein Data Bank (PDB) codes, secondary structure and solvent accessibility of residues at mutant positions, measuring methods, experimental conditions, such as buffers, ions and additives, and literature information. A WWW interface facilitates users to search data based on various conditions, feasibility to select the terms for output and different sorting options. Further, PINT is cross-linked with other related databases, PIR, SWISS-PROT, PDB and NCBI PUBMED literature database. The database is freely available at http://www.bioinfodatabase.com/pint/index.html. PMID:16381844

  6. Biochemical Approaches for Discovering Protein-Protein Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions or protein complexes are indigenous to nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For ex...

  7. Domains mediate protein-protein interactions and nucleate protein assemblies.

    PubMed

    Costa, S; Cesareni, G

    2008-01-01

    Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions. PMID:18491061

  8. Bacteriophage Protein–Protein Interactions

    PubMed Central

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage–host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  9. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-10-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3. PMID:26510391

  10. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  11. Split-Protein Systems: Beyond Binary Protein-Protein Interactions

    PubMed Central

    Shekhawat, Sujan S.; Ghosh, Indraneel

    2011-01-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome [1], a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, E. coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  12. Split-protein systems: beyond binary protein-protein interactions.

    PubMed

    Shekhawat, Sujan S; Ghosh, Indraneel

    2011-12-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  13. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  14. Energy design for protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Ravikant, D. V. S.; Elber, Ron

    2011-08-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions.

  15. Interactions of Pathological Hallmark Proteins

    PubMed Central

    Oláh, Judit; Vincze, Orsolya; Virók, Dezső; Simon, Dóra; Bozsó, Zsolt; Tőkési, Natália; Horváth, István; Hlavanda, Emma; Kovács, János; Magyar, Anna; Szűcs, Mária; Orosz, Ferenc; Penke, Botond; Ovádi, Judit

    2011-01-01

    The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ42 tightly bound to TPPP/p25 (Kd = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ42, α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease. PMID:21832049

  16. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  17. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  18. Hydrodynamic interactions in protein folding

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Niewieczerzał, Szymon

    2009-03-01

    We incorporate hydrodynamic interactions (HIs) in a coarse-grained and structure-based model of proteins by employing the Rotne-Prager hydrodynamic tensor. We study several small proteins and demonstrate that HIs facilitate folding. We also study HIV-1 protease and show that HIs make the flap closing dynamics faster. The HIs are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state.

  19. Hydrodynamic interactions in protein folding.

    PubMed

    Cieplak, Marek; Niewieczerzał, Szymon

    2009-03-28

    We incorporate hydrodynamic interactions (HIs) in a coarse-grained and structure-based model of proteins by employing the Rotne-Prager hydrodynamic tensor. We study several small proteins and demonstrate that HIs facilitate folding. We also study HIV-1 protease and show that HIs make the flap closing dynamics faster. The HIs are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state. PMID:19334888

  20. Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation

    PubMed Central

    Dermody, Jessica L.; Dreyfuss, Jonathan M.; Villén, Judit; Ogundipe, Babatunde; Gygi, Steven P.; Park, Peter J.; Ponticelli, Alfred S.; Moore, Claire L.; Buratowski, Stephen; Bucheli, Miriam E.

    2008-01-01

    The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation. PMID:18818768

  1. The Role of Siglec-1 and SR-BI Interaction in the Phagocytosis of Oxidized Low Density Lipoprotein by Macrophages

    PubMed Central

    Li, Chang; Zhu, Lin; Wu, Li-juan; Zhong, Ren-qian

    2013-01-01

    Background Macrophages play a proatherosclerotic role in atherosclerosis via oxLDL uptake. As an adhesion molecular of I-type lectins, Siglec-1 is highly expressed on circulating monocytes and plaque macrophages of atherosclerotic patients, but the exact role of Siglec-1 has not been elucidated. Methods In this study, oxLDL was used to stimulate Siglec-1 and some oxLDL receptors (SR-BI, CD64, CD32B, LOX-1 and TLR-4) expression on bone marrow-derived macrophages, whereas small interfering RNA was used to down-regulate Siglec-1. Meanwhile, an ELISA-based assay for Siglec-1-oxLDL interaction was performed, and co-immunoprecipitation (co-IP) and laser scanning confocal microscopy (LSCM) were used to determine the role of Siglec-1 in oxLDL uptake by macrophages. Results We found that oxLDL could up-regulate the expression of various potential oxLDL receptors, including Siglec-1, in a dose-dependent manner. Moreover, down-regulation of Siglec-1 could attenuate oxLDL uptake by Oil red O staining. LSCM revealed that Siglec-1 and CD64/SR-BI may colocalize on oxLDL-stimulated macrophage surface, whereas co-IP showed that Siglec-1 and SR-BI can be immunoprecipitated by each other. However, no direct interaction between Siglec-1 and oxLDL was found in the in vitro protein interaction system. Conclusions Thus, Siglec-1 can interact with SR-BI in the phagocytosis of oxLDL by macrophages, rather than act as an independent receptor for oxLDL. PMID:23520536

  2. Protein-Inhibitor Interaction Studies Using NMR

    PubMed Central

    Ishima, Rieko

    2015-01-01

    Solution-state NMR has been widely applied to determine the three-dimensional structure, dynamics, and molecular interactions of proteins. The designs of experiments used in protein NMR differ from those used for small-molecule NMR, primarily because the information available prior to an experiment, such as molecular mass and knowledge of the primary structure, is unique for proteins compared to small molecules. In this review article, protein NMR for structural biology is introduced with comparisons to small-molecule NMR, such as descriptions of labeling strategies and the effects of molecular dynamics on relaxation. Next, applications for protein NMR are reviewed, especially practical aspects for protein-observed ligand-protein interaction studies. Overall, the following topics are described: (1) characteristics of protein NMR, (2) methods to detect protein-ligand interactions by NMR, and (3) practical aspects of carrying out protein-observed inhibitor-protein interaction studies. PMID:26361636

  3. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    PubMed Central

    Sun, Zheng; Xiang, Jianhai

    2014-01-01

    WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1) and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP) encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA), two integrin beta (ITGB), and one syndecan (SDC). Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp. PMID:24982879

  4. Subcellular and subnuclear distribution of high-light responsive serine/arginine-rich proteins, atSR45a and atSR30, in Arabidopsis thaliana.

    PubMed

    Mori, Tatsuya; Yoshimura, Kazuya; Nosaka, Ryota; Sakuyama, Harumi; Koike, Yoshiyuki; Tanabe, Noriaki; Maruta, Takanori; Tamoi, Masahiro; Shigeoka, Shigeru

    2012-01-01

    Here, we demonstrated the involvement of the domains in Arabidopsis high-light responsive serine/arginine-rich (SR) and SR-like proteins, atSR30 and atSR45a, respectively, in subcellular and subnuclear distribution using a series of structural domain-deleted mutants. Judging from the localization of the transiently expressed domain-deleted mutants in onion epidermal cells, the C terminal low complexity domain rich in arginine-serine repeats (C-RS) domain of atSR30 appeared to be necessary for the nuclear localization. On the other hand, the N-terminal RS (N-RS) domain of atSR45a was necessary for the accurate nuclear localization, although the N- or C-RS domain alone was sufficient for the nuclear speckled organization. The phosphorylation of RS domains of atSR45a is irrelevant to the regulation of its localization. atSR45a and atSR30 were co-localized in the speckles, suggesting their collaborative roles in the regulation of alternative splicing events. PMID:23132568

  5. Proteins interacting with cloning scars: a source of false positive protein-protein interactions

    PubMed Central

    Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

    2015-01-01

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

  6. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  7. Protein-protein interactions in DNA mismatch repair.

    PubMed

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  8. RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

    PubMed Central

    Buratti, Emanuele; Muro, Andrés F.; Giombi, Maurizio; Gherbassi, Daniel; Iaconcig, Alessandra; Baralle, Francisco E.

    2004-01-01

    In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing. PMID:14729981

  9. Transient protein-protein interactions visualized by solution NMR.

    PubMed

    Liu, Zhu; Gong, Zhou; Dong, Xu; Tang, Chun

    2016-01-01

    Proteins interact with each other to establish their identities in cell. The affinities for the interactions span more than ten orders of magnitude, and KD values in μM-mM regimen are considered transient and are important in cell signaling. Solution NMR including diamagnetic and paramagnetic techniques has enabled atomic-resolution depictions of transient protein-protein interactions. Diamagnetic NMR allows characterization of protein complexes with KD values up to several mM, whereas ultraweak and fleeting complexes can be modeled with the use of paramagnetic NMR especially paramagnetic relaxation enhancement (PRE). When tackling ever-larger protein complexes, PRE can be particularly useful in providing long-range intermolecular distance restraints. As NMR measurements are averaged over the ensemble of complex structures, structural information for dynamic protein-protein interactions besides the stereospecific one can often be extracted. Herein the protein interaction dynamics are exemplified by encounter complexes, alternative binding modes, and coupled binding/folding of intrinsically disordered proteins. Further integration of NMR with other biophysical techniques should allow better visualization of transient protein-protein interactions. In particular, single-molecule data may facilitate the interpretation of ensemble-averaged NMR data. Though same structures of proteins and protein complexes were found in cell as in diluted solution, we anticipate that the dynamics of transient protein protein-protein interactions be different, which awaits awaits exploration by NMR. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:25896389

  10. How do oncoprotein mutations rewire protein-protein interaction networks?

    PubMed

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  11. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

    PubMed

    Ray, Kausik

    2015-01-01

    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  12. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

  13. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  14. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  15. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe” refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  16. How many protein-protein interactions types exist in nature?

    PubMed

    Garma, Leonardo; Mukherjee, Srayanta; Mitra, Pralay; Zhang, Yang

    2012-01-01

    "Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  17. Computational drug design targeting protein-protein interactions.

    PubMed

    Bienstock, Rachelle J

    2012-01-01

    Novel discoveries in molecular disease pathways within the cell, combined with increasing information regarding protein binding partners has lead to a new approach in drug discovery. There is interest in designing drugs to modulate protein-protein interactions as opposed to solely targeting the catalytic active site within a single enzyme or protein. There are many challenges in this new approach to drug discovery, particularly since the protein-protein interface has a larger surface area, can comprise a discontinuous epitope, and is more amorphous and less well defined than the typical drug design target, a small contained enzyme-binding pocket. Computational methods to predict modes of protein-protein interaction, as well as protein interface hot spots, have garnered significant interest, in order to facilitate the development of drugs to successfully disrupt and inhibit protein-protein interactions. This review summarizes some current methods available for computational protein-protein docking, as well as tabulating some examples of the successful design of antagonists and small molecule inhibitors for protein-protein interactions. Several of these drugs are now beginning to appear in the clinic. PMID:22316151

  18. Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.

    PubMed

    Zhang, Xiao-Ning; Mount, Stephen M

    2009-07-01

    The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3' splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions. PMID:19403727

  19. Current Experimental Methods for Characterizing Protein-Protein Interactions.

    PubMed

    Zhou, Mi; Li, Qing; Wang, Renxiao

    2016-04-19

    Protein molecules often interact with other partner protein molecules in order to execute their vital functions in living organisms. Characterization of protein-protein interactions thus plays a central role in understanding the molecular mechanism of relevant protein molecules, elucidating the cellular processes and pathways relevant to health or disease for drug discovery, and charting large-scale interaction networks in systems biology research. A whole spectrum of methods, based on biophysical, biochemical, or genetic principles, have been developed to detect the time, space, and functional relevance of protein-protein interactions at various degrees of affinity and specificity. This article presents an overview of these experimental methods, outlining the principles, strengths and limitations, and recent developments of each type of method. PMID:26864455

  20. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  1. Solid state NMR and protein-protein interactions in membranes.

    PubMed

    Miao, Yimin; Cross, Timothy A

    2013-12-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins. PMID:24034903

  2. Measuring protein interactions by microchip self-interaction chromatography.

    PubMed

    García, Carlos D; Hadley, DeGail J; Wilson, W William; Henry, Charles S

    2003-01-01

    The self-interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self-interaction chromatography has the potential to provide a quantitative method for determination of protein self-interactions amenable to high-throughput screening. This paper describes the construction and characterization of a microchip separation system for low-pressure self-interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile-phase composition, amount of protein injected, flow rate, and stationary-phase modification. The capacity factors (k') as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B(22)) obtained by static light scattering, showing the ability of the chip to accurately determine protein-protein interactions. A 500-fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein-protein interactions. PMID:12790668

  3. A Microfluidic Platform for Characterization of Protein-Protein Interactions.

    PubMed

    Javanmard, Mehdi; Talasaz, Amirali H; Nemat-Gorgani, Mohsen; Huber, David E; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W

    2009-08-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  4. The Intrinsic Geometric Structure of Protein-Protein Interaction Networks for Protein Interaction Prediction.

    PubMed

    Fang, Yi; Sun, Mengtian; Dai, Guoxian; Ramain, Karthik

    2016-01-01

    Recent developments in high-throughput technologies for measuring protein-protein interaction (PPI) have profoundly advanced our ability to systematically infer protein function and regulation. However, inherently high false positive and false negative rates in measurement have posed great challenges in computational approaches for the prediction of PPI. A good PPI predictor should be 1) resistant to high rate of missing and spurious PPIs, and 2) robust against incompleteness of observed PPI networks. To predict PPI in a network, we developed an intrinsic geometry structure (IGS) for network, which exploits the intrinsic and hidden relationship among proteins in network through a heat diffusion process. In this process, all explicit PPIs participate simultaneously to glue local infinitesimal and noisy experimental interaction data to generate a global macroscopic descriptions about relationships among proteins. The revealed implicit relationship can be interpreted as the probability of two proteins interacting with each other. The revealed relationship is intrinsic and robust against individual, local and explicit protein interactions in the original network. We apply our approach to publicly available PPI network data for the evaluation of the performance of PPI prediction. Experimental results indicate that, under different levels of the missing and spurious PPIs, IGS is able to robustly exploit the intrinsic and hidden relationship for PPI prediction with a higher sensitivity and specificity compared to that of recently proposed methods. PMID:26886733

  5. Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S.

    PubMed

    Gouldson, P; Legoux, P; Carillon, C; Dumont, X; Le Fur, G; Ferrara, P; Shire, D

    2000-02-18

    We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor. PMID:10688974

  6. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949

  7. Protein interaction networks from literature mining

    NASA Astrophysics Data System (ADS)

    Ihara, Sigeo

    2005-03-01

    The ability to accurately predict and understand physiological changes in the biological network system in response to disease or drug therapeutics is of crucial importance in life science. The extensive amount of gene expression data generated from even a single microarray experiment often proves difficult to fully interpret and comprehend the biological significance. An increasing knowledge of protein interactions stored in the PubMed database, as well as the advancement of natural language processing, however, makes it possible to construct protein interaction networks from the gene expression information that are essential for understanding the biological meaning. From the in house literature mining system we have developed, the protein interaction network for humans was constructed. By analysis based on the graph-theoretical characterization of the total interaction network in literature, we found that the network is scale-free and semantic long-ranged interactions (i.e. inhibit, induce) between proteins dominate in the total interaction network, reducing the degree exponent. Interaction networks generated based on scientific text in which the interaction event is ambiguously described result in disconnected networks. In contrast interaction networks based on text in which the interaction events are clearly stated result in strongly connected networks. The results of protein-protein interaction networks obtained in real applications from microarray experiments are discussed: For example, comparisons of the gene expression data indicative of either a good or a poor prognosis for acute lymphoblastic leukemia with MLL rearrangements, using our system, showed newly discovered signaling cross-talk.

  8. Protein-protein interactions and genetic diseases: The Interactome

    PubMed Central

    Lage, Kasper

    2014-01-01

    Protein-protein interactions mediate essentially all biological processes. Despite the quality of these data being widely questioned a decade ago, the reproducibility of large-scale protein interaction data is now much improved and there is little question that the latest screens are of high quality. Moreover, common data standards and coordinated curation practices between the databases that collect the interactions have made these valuable data available to a wide group of researchers. Here, I will review how protein-protein interactions are measured, collected and quality controlled. I discuss how the architecture of molecular protein networks have informed disease biology, and how these data are now being computationally integrated with the newest genomic technologies, in particular genome-wide association studies and exome-sequencing projects, to improve our understanding of molecular processes perturbed by genetics in human diseases. PMID:24892209

  9. APID: Agile Protein Interaction DataAnalyzer.

    PubMed

    Prieto, Carlos; De Las Rivas, Javier

    2006-07-01

    Agile Protein Interaction DataAnalyzer (APID) is an interactive bioinformatics web tool developed to integrate and analyze in a unified and comparative platform main currently known information about protein-protein interactions demonstrated by specific small-scale or large-scale experimental methods. At present, the application includes information coming from five main source databases enclosing an unified sever to explore >35 000 different proteins and 111 000 different proven interactions. The web includes search tools to query and browse upon the data, allowing selection of the interaction pairs based in calculated parameters that weight and qualify the reliability of each given protein interaction. Such parameters are for the 'proteins': connectivity, cluster coefficient, Gene Ontology (GO) functional environment, GO environment enrichment; and for the 'interactions': number of methods, GO overlapping, iPfam domain-domain interaction. APID also includes a graphic interactive tool to visualize selected sub-networks and to navigate on them or along the whole interaction network. The application is available open access at http://bioinfow.dep.usal.es/apid/. PMID:16845013

  10. An Interactive Introduction to Protein Structure

    ERIC Educational Resources Information Center

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  11. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  12. Noninvasive imaging of protein-protein interactions in living animals

    NASA Astrophysics Data System (ADS)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  13. DIP: The Database of Interacting Proteins

    DOE Data Explorer

    The DIP Database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent set of protein-protein interactions. By interaction, the DIP Database creators mean that two amino acid chains were experimentally identified to bind to each other. The database lists such pairs to aid those studying a particular protein-protein interaction but also those investigating entire regulatory and signaling pathways as well as those studying the organisation and complexity of the protein interaction network at the cellular level. The data stored within the DIP database were curated, both, manually by expert curators and also automatically using computational approaches that utilize the knowledge about the protein-protein interaction networks extracted from the most reliable, core subset of the DIP data. It is a relational database that can be searched by protein, sequence, motif, article information, and pathBLAST. The website also serves as an access point to a number of projects related to DIP, such as LiveDIP, The Database of Ligand-Receptor Partners (DLRP) and JDIP. Users have free and open access to DIP after login. [Taken from the DIP Guide and the DIP website] (Specialized Interface) (Registration Required)

  14. Predicting Physical Interactions between Protein Complexes*

    PubMed Central

    Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

    2013-01-01

    Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

  15. Evolutionarily Conserved Herpesviral Protein Interaction Networks

    PubMed Central

    Fossum, Even; Friedel, Caroline C.; Rajagopala, Seesandra V.; Titz, Björn; Baiker, Armin; Schmidt, Tina; Kraus, Theo; Stellberger, Thorsten; Rutenberg, Christiane; Suthram, Silpa; Bandyopadhyay, Sourav; Rose, Dietlind; von Brunn, Albrecht; Uhlmann, Mareike; Zeretzke, Christine; Dong, Yu-An; Boulet, Hélène; Koegl, Manfred; Bailer, Susanne M.; Koszinowski, Ulrich; Ideker, Trey; Uetz, Peter; Zimmer, Ralf; Haas, Jürgen

    2009-01-01

    Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species. PMID:19730696

  16. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

    PubMed Central

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M.; Brandl, Holger; Schwich, Oliver D.; Steiner, Michaela C.; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M.

    2016-01-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends. PMID:26944680

  17. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    PubMed

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  18. PLIC: protein-ligand interaction clusters.

    PubMed

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein- ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of ∼68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of

  19. PPIM: A Protein-Protein Interaction Database for Maize.

    PubMed

    Zhu, Guanghui; Wu, Aibo; Xu, Xin-Jian; Xiao, Pei-Pei; Lu, Le; Liu, Jingdong; Cao, Yongwei; Chen, Luonan; Wu, Jun; Zhao, Xing-Ming

    2016-02-01

    Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop. PMID:26620522

  20. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  1. Capturing the Interaction Potential of Amyloidogenic Proteins

    SciTech Connect

    Javid, Nadeem; Vogtt, Karsten; Winter, Roland; Krywka, Christina; Tolan, Metin

    2007-07-13

    Experimentally derived static structure factors obtained for the aggregation-prone protein insulin were analyzed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek potential. The data reveal that the protein self-assembles into equilibrium clusters already at low concentrations. Furthermore, striking differences regarding interaction forces between aggregation-prone proteins such as insulin in the preaggregated regime and natively stable globular proteins are found.

  2. Methods for analyzing and quantifying protein-protein interaction.

    PubMed

    Syafrizayanti; Betzen, Christian; Hoheisel, Jörg D; Kastelic, Damjana

    2014-02-01

    Genome sequencing has led to the identification of many proteins, which had not been recognized before. In consequence, the basic set of human proteins is generally known. Far less information, however, exists about protein-protein interactions, which are required and responsible for cellular activities and their control. Many protein isoforms that result from mutations, splice-variations and post-translational modifications also come into play. Until recently, interactions of only few protein partners could be analyzed in a single experiment. However, this does not meet the challenge of investigating the highly complex interaction patterns in cellular systems. It is made even more demanding by the need to determine the intensity of interactions quantitatively in order to properly understand protein interplay. Currently available techniques vary with respect to accuracy, reliability, reproducibility and throughput and their performances range from a mere qualitative demonstration of binding to a quantitative characterization of affinities. In this article, an overview is given of the methodologies available for analysis of protein-protein interactions. PMID:24393018

  3. Signature Product Code for Predicting Protein-Protein Interactions

    SciTech Connect

    Martin, Shawn B.; Brown, William M.

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictions about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.

  4. Signature Product Code for Predicting Protein-Protein Interactions

    Energy Science and Technology Software Center (ESTSC)

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictionsmore » about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.« less

  5. Quantitative interaction proteomics of neurodegenerative disease proteins.

    PubMed

    Hosp, Fabian; Vossfeldt, Hannes; Heinig, Matthias; Vasiljevic, Djordje; Arumughan, Anup; Wyler, Emanuel; Landthaler, Markus; Hubner, Norbert; Wanker, Erich E; Lannfelt, Lars; Ingelsson, Martin; Lalowski, Maciej; Voigt, Aaron; Selbach, Matthias

    2015-05-19

    Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD. PMID:25959826

  6. Protein interaction mapping: A Drosophila case study

    PubMed Central

    Formstecher, Etienne; Aresta, Sandra; Collura, Vincent; Hamburger, Alexandre; Meil, Alain; Trehin, Alexandra; Reverdy, Céline; Betin, Virginie; Maire, Sophie; Brun, Christine; Jacq, Bernard; Arpin, Monique; Bellaiche, Yohanns; Bellusci, Saverio; Benaroch, Philippe; Bornens, Michel; Chanet, Roland; Chavrier, Philippe; Delattre, Olivier; Doye, Valérie; Fehon, Richard; Faye, Gérard; Galli, Thierry; Girault, Jean-Antoine; Goud, Bruno; de Gunzburg, Jean; Johannes, Ludger; Junier, Marie-Pierre; Mirouse, Vincent; Mukherjee, Ashim; Papadopoulo, Dora; Perez, Franck; Plessis, Anne; Rossé, Carine; Saule, Simon; Stoppa-Lyonnet, Dominique; Vincent, Alain; White, Michael; Legrain, Pierre; Wojcik, Jérôme; Camonis, Jacques; Daviet, Laurent

    2005-01-01

    The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps. PMID:15710747

  7. Multilevel regulation of protein protein interactions in biological circuitry

    NASA Astrophysics Data System (ADS)

    Beckett, Dorothy

    2005-06-01

    Protein-protein interactions are central to biology and, in this 'post-genomic era', prediction of these interactions has become the goal of many computational efforts. Close inspection of even relatively simple biological regulatory circuitry reveals multiple levels of control of the contributing protein interactions. The fundamental probability that an interaction will occur under a given set of conditions is difficult to predict because the relationship between structure and energy is not known. Layered on this basic difficulty are allosteric control mechanisms involving post-translational modification or small ligand binding. In addition, many biological processes involve multiple protein-protein interactions, some of which may be cooperative or even competitive. Finally, although the emphasis in predicting protein interactions is based on equilibrium thermodynamic principles, kinetics can be a major controlling feature in these systems. This complexity reinforces the necessity of performing detailed quantitative studies of the component interactions of complex biological regulatory systems. Results of such studies will help us to bridge the gap between our knowledge of structure and our understanding of functional biology.

  8. Blue copper proteins: Synthesis, spectra, and structures of CuIN3(SR) and CuIIN3(SR) active site analogues

    PubMed Central

    Thompson, Jeffery S.; Marks, Tobin J.; Ibers, James A.

    1977-01-01

    The reaction of Cu(SR) or [Cu(SR)][ClO4] derivatives (SR = p-nitrobenzenethiolate or O-ethylcysteinate) with potassium hydrotris(3,5-dimethyl-1-pyrazolyl)borate produces redox pairs of the stoichiometry CuIN3(SR) and CuIIN3(SR). These complexes are well-defined synthetic approximations to the proposed N3S binding sites of blue (type 1) copper electron transfer proteins. The compounds were investigated by a variety of chemical and spectral (optical, resonance Raman, and electron paramagnetic resonance) techniques; the complex K[Cu(hydrotris(3,5-dimethyl-1-pyrazolyl)borate)(p- NO2C6H4S]-2 acetone was also studied by single-crystal x-ray diffraction methods. The spectrochemical characteristics of the CuIIN3(SR) species are in large part similar to the native system and thus provide some perspective regarding the origin of the unique type 1 spectral parameters and electron transfer properties. PMID:16592426

  9. Characterizing carbohydrate-protein interactions by NMR

    PubMed Central

    Bewley, Carole A.; Shahzad-ul-Hussan, Syed

    2013-01-01

    Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792

  10. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  11. Van der Waals interactions involving proteins.

    PubMed Central

    Roth, C M; Neal, B L; Lenhoff, A M

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth. Images FIGURE 3 PMID:8789115

  12. FAK and p53 protein interactions.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2011-09-01

    Focal Adhesion Kinase plays a major role in cell adhesion, motility, survival, proliferation, metastasis, angiogenesis and lymphangiogenesis. In 2004, we have cloned the promoter sequence of FAK and found that p53 inhibits its activity (BBA, v. 1678, 2004). In 2005, we were the first group to show that FAK and p53 proteins directly interact in the cells (JBC, v. 280, 2005). We have shown that FAK and p53 proteins interact in the cytoplasm and in the nucleus by immunoprecipitation, pull-down and confocal microscopy assays. We have shown that FAK inhibited activity of p53 with the transcriptional targets: p21, Bax and Mdm-2 through protein-protein interactions. We identified the 7 amino-acid site in p53 that is involved in interaction with FAK protein. The present review will discuss the interaction of FAK and p53 proteins and discuss the mechanism of FAK-p53 loop regulation: inhibition of FAK promoter activity by p53 protein and also inhibition of p53 transcriptional activity by FAK protein. PMID:21355845

  13. RNA Protein Interaction in Neurons

    PubMed Central

    Darnell, Robert B.

    2013-01-01

    Neurons have their own systems for regulating RNA. Several multigene families encode RNA binding proteins (RNABPs) that are uniquely expressed in neurons, including the well-known neuron-specific markers ELAV and NeuN, and the disease antigen NOVA. New technologies have emerged in recent years to assess the function of these proteins in vivo, and the answers are yielding insights into how and why neurons may regulate RNA in special ways—to increase cellular complexity, to spatially localize mRNA, and to regulate their expression in response to synaptic stimuli. The functions of such restricted neuronal proteins is likely to be complimented by more widely expressed RNABPs that may themselves have developed specialized functions in neurons, including Argonaute/miRNAs. Here we review what is known about such RNABPs, and explore the potential biologic and neurologic significance of neuronal RNA regulatory systems. PMID:23701460

  14. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  15. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. PMID:21377472

  16. Interface-Resolved Network of Protein-Protein Interactions

    PubMed Central

    Johnson, Margaret E.; Hummer, Gerhard

    2013-01-01

    We define an interface-interaction network (IIN) to capture the specificity and competition between protein-protein interactions (PPI). This new type of network represents interactions between individual interfaces used in functional protein binding and thereby contains the detail necessary to describe the competition and cooperation between any pair of binding partners. Here we establish a general framework for the construction of IINs that merges computational structure-based interface assignment with careful curation of available literature. To complement limited structural data, the inclusion of biochemical data is critical for achieving the accuracy and completeness necessary to analyze the specificity and competition between the protein interactions. Firstly, this procedure provides a means to clarify the information content of existing data on purported protein interactions and to remove indirect and spurious interactions. Secondly, the IIN we have constructed here for proteins involved in clathrin-mediated endocytosis (CME) exhibits distinctive topological properties. In contrast to PPI networks with their global and relatively dense connectivity, the fragmentation of the IIN into distinctive network modules suggests that different functional pressures act on the evolution of its topology. Large modules in the IIN are formed by interfaces sharing specificity for certain domain types, such as SH3 domains distributed across different proteins. The shared and distinct specificity of an interface is necessary for effective negative and positive design of highly selective binding targets. Lastly, the organization of detailed structural data in a network format allows one to identify pathways of specific binding interactions and thereby predict effects of mutations at specific surfaces on a protein and of specific binding inhibitors, as we explore in several examples. Overall, the endocytosis IIN is remarkably complex and rich in features masked in the coarser

  17. The Predicted Spectrum and Singlet-Triplet Interaction of the Hypermetallic Molecule SrOSr

    NASA Astrophysics Data System (ADS)

    Ostojić, B.; Jensen, Per; Schwerdtfeger, P.; Bunker, P. R.

    2013-10-01

    In accordance with previous studies in our group on Be, Mg, and Ca hypermetallic oxides, we find that SrOSr has a linear X-1Σg+ ground electronic state and a very low lying first excited -3Σu+ triplet electronic state. No gas-phase spectrum of this molecule has been assigned yet, and to encourage and assist in its discovery we present a complete ab initio simulation, with absolute intensities, of the infrared absorption spectrum for both electronic states. The three-dimensional potential energy surfaces and the electric dipole moment surfaces of the X-1Σg+ and -3Σu+ electronic states are calculated using a multireference configuration interaction (MRCISD) approach in combination with internally contracted multireference perturbation theory (RS2C) based on complete active space self-consistent field (CASSCF) wave functions applying a Sadlej pVTZ basis set for both O and Sr and the Stuttgart relativistic small-core effective core potential for Sr. The infrared spectra are simulated using the MORBID program system. We also calculate vertical excitation energies and transition moments for several excited singlet and triplet electronic states in order to predict the positions and intensities of the most prominent singlet and triplet electronic absorption bands. Finally, for this heavy molecule, we calculate the singlet-triplet interaction matrix elements between close-lying vibronic levels of the X- and - electronic states and find them to be very small.

  18. Moonlighting proteins in sperm-egg interactions.

    PubMed

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins. PMID:25399599

  19. Website on Protein Interaction and Protein Structure Related Work

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  20. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

    PubMed

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  1. Structure and magnetic interactions in (Sr, Sb)-doped lanthanum manganites

    NASA Astrophysics Data System (ADS)

    Karpinsky, D. V.; Troyanchuk, I. O.; Silibin, M. V.; Gavrilov, S. A.; Bushinky, M. V.; Sikolenko, V.; Frontzek, M.

    2016-05-01

    Ceramic samples La1-2xSr2xMn1-xSbxO3 (x≤0.2) have been studied by X-ray and neutron powder diffraction, magnetization measurements and charge density calculations have also been performed. The compounds are characterized by the rhombohedral structure at room temperature; temperature decrease causes structural transition to the orthorhombic structure. La1-2xSr2xMn1-xSbxO3 ceramics are characterized by homovalent manganese ions and have long range ferromagnetic order which gradually diminishes with the chemical substitution. It is considered that magnetic properties of the compounds are determined by the dominance of ferromagnetic superexchange interactions stabilized in the orbital disordered orthorhombic phase. Significant covalent component of the Mn-O chemical bonds contributes to the long-range ferromagnetic order of the compounds, the covalency of the chemical bonds is assumed to be isotropically distributed over the orthorhombic phase.

  2. Novel computational methods to design protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Zhou, Alice Qinhua; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite the abundance of structural data, we still cannot accurately predict the structural and energetic changes resulting from mutations at protein interfaces. The inadequacy of current computational approaches to the analysis and design of protein-protein interactions has hampered the development of novel therapeutic and diagnostic agents. In this work, we apply a simple physical model that includes only a minimal set of geometrical constraints, excluded volume, and attractive van der Waals interactions to 1) rank the binding affinity of mutants of tetratricopeptide repeat proteins with their cognate peptides, 2) rank the energetics of binding of small designed proteins to the hydrophobic stem region of the influenza hemagglutinin protein, and 3) predict the stability of T4 lysozyme and staphylococcal nuclease mutants. This work will not only lead to a fundamental understanding of protein-protein interactions, but also to the development of efficient computational methods to rationally design protein interfaces with tunable specificity and affinity, and numerous applications in biomedicine. NSF DMR-1006537, PHY-1019147, Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, and Howard Hughes Medical Institute.

  3. Systematic computational prediction of protein interaction networks.

    PubMed

    Lees, J G; Heriche, J K; Morilla, I; Ranea, J A; Orengo, C A

    2011-06-01

    Determining the network of physical protein associations is an important first step in developing mechanistic evidence for elucidating biological pathways. Despite rapid advances in the field of high throughput experiments to determine protein interactions, the majority of associations remain unknown. Here we describe computational methods for significantly expanding protein association networks. We describe methods for integrating multiple independent sources of evidence to obtain higher quality predictions and we compare the major publicly available resources available for experimentalists to use. PMID:21572181

  4. Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

    PubMed Central

    Khor, Susan

    2014-01-01

    Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

  5. Regulation of alternative splicing of CD45 by antagonistic effects of SR protein splicing factors.

    PubMed

    ten Dam, G B; Zilch, C F; Wallace, D; Wieringa, B; Beverley, P C; Poels, L G; Screaton, G R

    2000-05-15

    CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable exons (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express exon A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation. PMID:10799890

  6. Teaching Noncovalent Interactions Using Protein Molecular Evolution

    ERIC Educational Resources Information Center

    Fornasari, Maria Silvina; Parisi, Gustavo; Echave, Julian

    2008-01-01

    Noncovalent interactions and physicochemical properties of amino acids are important topics in biochemistry courses. Here, we present a computational laboratory where the capacity of each of the 20 amino acids to maintain different noncovalent interactions are used to investigate the stabilizing forces in a set of proteins coming from organisms…

  7. Evolving new protein-protein interaction specificity through promiscuous intermediates.

    PubMed

    Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N; Perchuk, Barrett S; Crosson, Sean; Laub, Michael T

    2015-10-22

    Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution. PMID:26478181

  8. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  9. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    SciTech Connect

    Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.; Sandri-Goldin, Rozanne M.

    2010-06-05

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  10. A Protein Interaction Map of Drosophila melanogaster

    NASA Astrophysics Data System (ADS)

    Giot, L.; Bader, J. S.; Brouwer, C.; Chaudhuri, A.; Kuang, B.; Li, Y.; Hao, Y. L.; Ooi, C. E.; Godwin, B.; Vitols, E.; Vijayadamodar, G.; Pochart, P.; Machineni, H.; Welsh, M.; Kong, Y.; Zerhusen, B.; Malcolm, R.; Varrone, Z.; Collis, A.; Minto, M.; Burgess, S.; McDaniel, L.; Stimpson, E.; Spriggs, F.; Williams, J.; Neurath, K.; Ioime, N.; Agee, M.; Voss, E.; Furtak, K.; Renzulli, R.; Aanensen, N.; Carrolla, S.; Bickelhaupt, E.; Lazovatsky, Y.; DaSilva, A.; Zhong, J.; Stanyon, C. A.; Finley, R. L.; White, K. P.; Braverman, M.; Jarvie, T.; Gold, S.; Leach, M.; Knight, J.; Shimkets, R. A.; McKenna, M. P.; Chant, J.; Rothberg, J. M.

    2003-12-01

    Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

  11. Computational Methods to Predict Protein Interaction Partners

    NASA Astrophysics Data System (ADS)

    Valencia, Alfonso; Pazos, Florencio

    In the new paradigm for studying biological phenomena represented by Systems Biology, cellular components are not considered in isolation but as forming complex networks of relationships. Protein interaction networks are among the first objects studied from this new point of view. Deciphering the interactome (the whole network of interactions for a given proteome) has been shown to be a very complex task. Computational techniques for detecting protein interactions have become standard tools for dealing with this problem, helping and complementing their experimental counterparts. Most of these techniques use genomic or sequence features intuitively related with protein interactions and are based on "first principles" in the sense that they do not involve training with examples. There are also other computational techniques that use other sources of information (i.e. structural information or even experimental data) or are based on training with examples.

  12. Protein interactions in concentrated ribonuclease solutions

    NASA Astrophysics Data System (ADS)

    Boyer, Mireille; Roy, Marie-Odile; Jullien, Magali; Bonneté, Françoise; Tardieu, Annette

    1999-01-01

    To investigate the protein interactions involved in the crystallization process of ribonuclease A, dynamic light scattering (DLS) and small angle X-ray scattering experiments (SAXS) were performed on concentrated solutions. Whereas the translational diffusion coefficient obtained from DLS is sensitive to thermodynamic and hydrodynamic interactions and permits to calculate an interaction parameter, the shape of the SAXS curves is related to the type of interaction (attractive or repulsive). We compared the effect of pH on protein interactions in the case of two types of crystallizing agents: a mixture of salts (3 M sodium chloride plus 0.2 M ammonium sulfate) and an organic solvent (ethanol). The results show that in the presence of ethanol, as in low salt, protein interactions become more attractive as the pH increases from 4 to 8 and approaches the isoelectric point. In contrast, a reverse effect is observed in high salt conditions: the strength of attractive interactions decreases as the pH increases. The range of the pH effect can be related to ionization of histidine residues, particularly those located in the active site of the protein. The present observations point out the important role played by localized charges in crystallization conditions, whatever the precipitating agent.

  13. Protein-protein interactions and prediction: a comprehensive overview.

    PubMed

    Sowmya, Gopichandran; Ranganathan, Shoba

    2014-01-01

    Molecular function in cellular processes is governed by protein-protein interactions (PPIs) within biological networks. Selective yet specific association of these protein partners contributes to diverse functionality such as catalysis, regulation, assembly, immunity, and inhibition in a cell. Therefore, understanding the principles of protein-protein association has been of immense interest for several decades. We provide an overview of the experimental methods used to determine PPIs and the key databases archiving this information. Structural and functional information of existing protein complexes confers knowledge on the principles of PPI, based on which a classification scheme for PPIs is then introduced. Obtaining high-quality non-redundant datasets of protein complexes for interaction characterisation is an essential step towards deciphering their underlying binding principles. Analysis of physicochemical features and their documentation has enhanced our understanding of the molecular basis of protein-protein association. We describe the diverse datasets created/collected by various groups and their key findings inferring distinguishing features. The currently available interface databases and prediction servers have also been compiled. PMID:23855658

  14. Inferring high-confidence human protein-protein interactions

    PubMed Central

    2012-01-01

    Background As numerous experimental factors drive the acquisition, identification, and interpretation of protein-protein interactions (PPIs), aggregated assemblies of human PPI data invariably contain experiment-dependent noise. Ascertaining the reliability of PPIs collected from these diverse studies and scoring them to infer high-confidence networks is a non-trivial task. Moreover, a large number of PPIs share the same number of reported occurrences, making it impossible to distinguish the reliability of these PPIs and rank-order them. For example, for the data analyzed here, we found that the majority (>83%) of currently available human PPIs have been reported only once. Results In this work, we proposed an unsupervised statistical approach to score a set of diverse, experimentally identified PPIs from nine primary databases to create subsets of high-confidence human PPI networks. We evaluated this ranking method by comparing it with other methods and assessing their ability to retrieve protein associations from a number of diverse and independent reference sets. These reference sets contain known biological data that are either directly or indirectly linked to interactions between proteins. We quantified the average effect of using ranked protein interaction data to retrieve this information and showed that, when compared to randomly ranked interaction data sets, the proposed method created a larger enrichment (~134%) than either ranking based on the hypergeometric test (~109%) or occurrence ranking (~46%). Conclusions From our evaluations, it was clear that ranked interactions were always of value because higher-ranked PPIs had a higher likelihood of retrieving high-confidence experimental data. Reducing the noise inherent in aggregated experimental PPIs via our ranking scheme further increased the accuracy and enrichment of PPIs derived from a number of biologically relevant data sets. These results suggest that using our high-confidence protein interactions

  15. Predicting protein-peptide interactions from scratch

    NASA Astrophysics Data System (ADS)

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin; Zou lab Team

    Protein-peptide interactions play an important role in many cellular processes. The ability to predict protein-peptide complex structures is valuable for mechanistic investigation and therapeutic development. Due to the high flexibility of peptides and lack of templates for homologous modeling, predicting protein-peptide complex structures is extremely challenging. Recently, we have developed a novel docking framework for protein-peptide structure prediction. Specifically, given the sequence of a peptide and a 3D structure of the protein, initial conformations of the peptide are built through protein threading. Then, the peptide is globally and flexibly docked onto the protein using a novel iterative approach. Finally, the sampled modes are scored and ranked by a statistical potential-based energy scoring function that was derived for protein-peptide interactions from statistical mechanics principles. Our docking methodology has been tested on the Peptidb database and compared with other protein-peptide docking methods. Systematic analysis shows significantly improved results compared to the performances of the existing methods. Our method is computationally efficient and suitable for large-scale applications. Nsf CAREER Award 0953839 (XZ) NIH R01GM109980 (XZ).

  16. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  17. Protein-protein interactions in reversibly assembled nanopatterns.

    PubMed

    Rakickas, Tomas; Gavutis, Martynas; Reichel, Annett; Piehler, Jacob; Liedberg, Bo; Valiokas, Ramūnas

    2008-10-01

    We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures. PMID:18788824

  18. Predicting protein-protein interactions based only on sequences information.

    PubMed

    Shen, Juwen; Zhang, Jian; Luo, Xiaomin; Zhu, Weiliang; Yu, Kunqian; Chen, Kaixian; Li, Yixue; Jiang, Hualiang

    2007-03-13

    Protein-protein interactions (PPIs) are central to most biological processes. Although efforts have been devoted to the development of methodology for predicting PPIs and protein interaction networks, the application of most existing methods is limited because they need information about protein homology or the interaction marks of the protein partners. In the present work, we propose a method for PPI prediction using only the information of protein sequences. This method was developed based on a learning algorithm-support vector machine combined with a kernel function and a conjoint triad feature for describing amino acids. More than 16,000 diverse PPI pairs were used to construct the universal model. The prediction ability of our approach is better than that of other sequence-based PPI prediction methods because it is able to predict PPI networks. Different types of PPI networks have been effectively mapped with our method, suggesting that, even with only sequence information, this method could be applied to the exploration of networks for any newly discovered protein with unknown biological relativity. In addition, such supplementary experimental information can enhance the prediction ability of the method. PMID:17360525

  19. Building protein interaction maps for Down's syndrome.

    PubMed

    Gardiner, Katheleen; Davisson, Muriel T; Crnic, Linda S

    2004-08-01

    Now that the complete sequences for human chromosome 21 and the orthologous mouse genomic regions are known, reasonably complete, conserved, protein-coding gene catalogues are also available. The central issue now facing Down's syndrome researchers is the correlation of increased expression of specific, normal, chromosome 21 genes with the development of specific deficits in learning and memory. Because of the number of candidate genes involved, the number of alternative splice variants of individual genes and the number of pathways in which these genes function, a pathway analysis approach will be critical to success. Here, three examples, both gene specific and pathway related, that would benefit from pathway analysis are discussed: (1) the potential roles of eight chromosome 21 proteins in RNA processing pathways; (2) the chromosome 21 protein intersectin 1 and its domain composition, alternative splicing, protein interactions and functions; and (3) the interactions of ten chromosome 21 proteins with components of the mitogen-activated protein kinase and the calcineurin signalling pathways. A productive approach to developing gene-phenotype correlations in Down's syndrome will make use of known and predicted functions and interactions of chromosome 21 genes to predict pathways that may be perturbed by their increased levels of expression. Investigations may then be targeted in animal models to specific interactions, intermediate steps or end-points of such pathways and the downstream - perhaps amplified - consequences of gene dosage directly assessed. Once pathway perturbations have been identified, the potential for rational design of therapeutics becomes practical. PMID:15355596

  20. Interaction prediction using conserved network motifs in protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Albert, Reka

    2005-03-01

    High-throughput protein interaction detection methods are strongly affected by false positive and false negative results. Focused experiments are needed to complement the large-scale methods by validating previously detected interactions but it is often difficult to decide which proteins to probe as interaction partners. Developing reliable computational methods assisting this decision process is a pressing need in bioinformatics. This talk will describe the recent developments in analyzing and understanding protein interaction networks, then present a method that uses the conserved properties of the protein network to identify and validate interaction candidates. We apply a number of machine learning algorithms to the protein connectivity information and achieve a surprisingly good overall performance in predicting interacting proteins. Using a ``leave-one-ou approach we find average success rates between 20-50% for predicting the correct interaction partner of a protein. We demonstrate that the success of these methods is based on the presence of conserved interaction motifs within the network. A reference implementation and a table with candidate interacting partners for each yeast protein are available at http://www.protsuggest.org

  1. Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

    SciTech Connect

    Curtis, R.A.; Blanch, H.W.; Prausnitz, J.M.

    1998-01-05

    Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

  2. The Host Proteins Transportin SR2/TNPO3 and Cyclophilin A Exert Opposing Effects on HIV-1 Uncoating

    PubMed Central

    Shah, Vaibhav B.; Shi, Jiong; Hout, David R.; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S.; Engelman, Alan

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  3. The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating.

    PubMed

    Shah, Vaibhav B; Shi, Jiong; Hout, David R; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S; Engelman, Alan; Aiken, Christopher

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  4. [Chemical libraries dedicated to protein-protein interactions].

    PubMed

    Sperandio, Olivier; Villoutreix, Bruno O; Morelli, Xavier; Roche, Philippe

    2015-03-01

    The identification of complete networks of protein-protein interactions (PPI) within a cell has contributed to major breakthroughs in understanding biological pathways, host-pathogen interactions and cancer development. As a consequence, PPI have emerged as a new class of promising therapeutic targets. However, they are still considered as a challenging class of targets for drug discovery programs. Recent successes have allowed the characterization of structural and physicochemical properties of protein-protein interfaces leading to a better understanding of how they can be disrupted with small molecule compounds. In addition, characterization of the profiles of PPI inhibitors has allowed the development of PPI-focused libraries. In this review, we present the current efforts at developing chemical libraries dedicated to these innovative targets. PMID:25855285

  5. KFC Server: interactive forecasting of protein interaction hot spots.

    PubMed

    Darnell, Steven J; LeGault, Laura; Mitchell, Julie C

    2008-07-01

    The KFC Server is a web-based implementation of the KFC (Knowledge-based FADE and Contacts) model-a machine learning approach for the prediction of binding hot spots, or the subset of residues that account for most of a protein interface's; binding free energy. The server facilitates the automated analysis of a user submitted protein-protein or protein-DNA interface and the visualization of its hot spot predictions. For each residue in the interface, the KFC Server characterizes its local structural environment, compares that environment to the environments of experimentally determined hot spots and predicts if the interface residue is a hot spot. After the computational analysis, the user can visualize the results using an interactive job viewer able to quickly highlight predicted hot spots and surrounding structural features within the protein structure. The KFC Server is accessible at http://kfc.mitchell-lab.org. PMID:18539611

  6. Prediction of protein-protein interactions based on protein-protein correlation using least squares regression.

    PubMed

    Huang, De-Shuang; Zhang, Lei; Han, Kyungsook; Deng, Suping; Yang, Kai; Zhang, Hongbo

    2014-01-01

    In order to transform protein sequences into the feature vectors, several works have been done, such as computing auto covariance (AC), conjoint triad (CT), local descriptor (LD), moran autocorrelation (MA), normalized moreaubroto autocorrelation (NMB) and so on. In this paper, we shall adopt these transformation methods to encode the proteins, respectively, where AC, CT, LD, MA and NMB are all represented by '+' in a unified manner. A new method, i.e. the combination of least squares regression with '+' (abbreviated as LSR(+)), will be introduced for encoding a protein-protein correlation-based feature representation and an interacting protein pair. Thus there are totally five different combinations for LSR(+), i.e. LSRAC, LSRCT, LSRLD, LSRMA and LSRNMB. As a result, we combined a support vector machine (SVM) approach with LSR(+) to predict protein-protein interactions (PPI) and PPI networks. The proposed method has been applied on four datasets, i.e. Saaccharomyces cerevisiae, Escherichia coli, Homo sapiens and Caenorhabditis elegans. The experimental results demonstrate that all LSR(+) methods outperform many existing representative algorithms. Therefore, LSR(+) is a powerful tool to characterize the protein-protein correlations and to infer PPI, whilst keeping high performance on prediction of PPI networks. PMID:25059329

  7. Annotation and retrieval in protein interaction databases

    NASA Astrophysics Data System (ADS)

    Cannataro, Mario; Hiram Guzzi, Pietro; Veltri, Pierangelo

    2014-06-01

    Biological databases have been developed with a special focus on the efficient retrieval of single records or the efficient computation of specialized bioinformatics algorithms against the overall database, such as in sequence alignment. The continuos production of biological knowledge spread on several biological databases and ontologies, such as Gene Ontology, and the availability of efficient techniques to handle such knowledge, such as annotation and semantic similarity measures, enable the development on novel bioinformatics applications that explicitly use and integrate such knowledge. After introducing the annotation process and the main semantic similarity measures, this paper shows how annotations and semantic similarity can be exploited to improve the extraction and analysis of biologically relevant data from protein interaction databases. As case studies, the paper presents two novel software tools, OntoPIN and CytoSeVis, both based on the use of Gene Ontology annotations, for the advanced querying of protein interaction databases and for the enhanced visualization of protein interaction networks.

  8. Transient DNA / RNA-protein interactions.

    PubMed

    Blanco, Francisco J; Montoya, Guillermo

    2011-05-01

    The great pace of biomolecular structure determination has provided a plethora of protein structures, but not as many structures of nucleic acids or of their complexes with proteins. The recognition of DNA and RNA molecules by proteins may produce large and relatively stable assemblies (such as the ribosome) or transient complexes (such as DNA clamps sliding through the DNA). These transient interactions are most difficult to characterize, but even in 'stable' complexes captured in crystal structures, the dynamics of the whole or part of the assembly pose great technical difficulties in understanding their function. The development and refinement of powerful experimental and computational tools have made it possible to learn a great deal about the relevance of these fleeting events for numerous biological processes. We discuss here the most recent findings and the challenges that lie ahead in the quest for a better understanding of protein-nucleic acid interactions. PMID:21410646

  9. Studying protein-protein interactions: progress, pitfalls and solutions.

    PubMed

    Hayes, Sheri; Malacrida, Beatrice; Kiely, Maeve; Kiely, Patrick A

    2016-08-15

    Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success. PMID:27528744

  10. Potential disruption of protein-protein interactions by graphene oxide.

    PubMed

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-14

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications. PMID:27306022

  11. Potential disruption of protein-protein interactions by graphene oxide

    NASA Astrophysics Data System (ADS)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-01

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  12. Peptiderive server: derive peptide inhibitors from protein-protein interactions.

    PubMed

    Sedan, Yuval; Marcu, Orly; Lyskov, Sergey; Schueler-Furman, Ora

    2016-07-01

    The Rosetta Peptiderive protocol identifies, in a given structure of a protein-protein interaction, the linear polypeptide segment suggested to contribute most to binding energy. Interactions that feature a 'hot segment', a linear peptide with significant binding energy compared to that of the complex, may be amenable for inhibition and the peptide sequence and structure derived from the interaction provide a starting point for rational drug design. Here we present a web server for Peptiderive, which is incorporated within the ROSIE web interface for Rosetta protocols. A new feature of the protocol also evaluates whether derived peptides are good candidates for cyclization. Fast computation times and clear visualization allow users to quickly assess the interaction of interest. The Peptiderive server is available for free use at http://rosie.rosettacommons.org/peptiderive. PMID:27141963

  13. A Method for Predicting Protein-Protein Interaction Types

    PubMed Central

    Silberberg, Yael

    2014-01-01

    Protein-protein interactions (PPIs) govern basic cellular processes through signal transduction and complex formation. The diversity of those processes gives rise to a remarkable diversity of interactions types, ranging from transient phosphorylation interactions to stable covalent bonding. Despite our increasing knowledge on PPIs in humans and other species, their types remain relatively unexplored and few annotations of types exist in public databases. Here, we propose the first method for systematic prediction of PPI type based solely on the techniques by which the interaction was detected. We show that different detection methods are better suited for detecting specific types. We apply our method to ten interaction types on a large scale human PPI dataset. We evaluate the performance of the method using both internal cross validation and external data sources. In cross validation, we obtain an area under receiver operating characteristic (ROC) curve ranging from 0.65 to 0.97 with an average of 0.84 across the predicted types. Comparing the predicted interaction types to external data sources, we obtained significant agreements for phosphorylation and ubiquitination interactions, with hypergeometric p-value = 2.3e−54 and 5.6e−28 respectively. We examine the biological relevance of our predictions using known signaling pathways and chart the abundance of interaction types in cell processes. Finally, we investigate the cross-relations between different interaction types within the network and characterize the discovered patterns, or motifs. We expect the resulting annotated network to facilitate the reconstruction of process-specific subnetworks and assist in predicting protein function or interaction. PMID:24625764

  14. Interaction of Sr-90 with site candidate soil for demonstration disposal facility at Serpong

    NASA Astrophysics Data System (ADS)

    Setiawan, Budi; Mila, Oktri; Safni

    2014-03-01

    Interaction of radiostrontium (Sr-90) with site candidate soil for demonstration disposal facility to be constructed in the near future at Serpong has been done. This activity is to anticipate the interim storage facility at Serpong nuclear area becomes full off condition, and show to the public how radioactive waste can be well managed with the existing technology. To ensure that the location is save, a reliability study of site candidate soil becomes very importance to be conducted through some experiments consisted some affected parameters such as contact time, effect of ionic strength, and effect of Sr+ ion in solution. Radiostrontium was used as a tracer on the experiments and has role as radionuclide reference in low-level radioactive waste due to its long half-live and it's easy to associate with organism in nature. So, interaction of radiostrontium and soil samples from site becomes important to be studied. Experiment was performed in batch method, and soil sample-solution containing radionuclide was mixed in a 20 ml of PE vial. Ratio of solid: liquid was 10-2 g/ml. Objective of the experiment is to collect the specific characteristics data of radionuclide sorption onto soil from site candidate. Distribution coefficient value was used as indicator where the amount of initial and final activities of radiostrontium in solution was compared. Result showed that equilibrium condition was reached after contact time 10 days with Kd values ranged from 1600-2350 ml/g. Increased in ionic strength in solution made decreased of Kd value into soil sample due to competition of background salt and radiostrontium into soil samples, and increased in Sr ion in solution caused decreased of Kd value in soil sample due to limitation of sorption capacity in soil samples. Fast condition in saturated of metal ion into soil samples was reached due to a simple reaction was occurred.

  15. Interaction of Sr-90 with site candidate soil for demonstration disposal facility at Serpong

    SciTech Connect

    Setiawan, Budi; Mila, Oktri; Safni

    2014-03-24

    Interaction of radiostrontium (Sr-90) with site candidate soil for demonstration disposal facility to be constructed in the near future at Serpong has been done. This activity is to anticipate the interim storage facility at Serpong nuclear area becomes full off condition, and show to the public how radioactive waste can be well managed with the existing technology. To ensure that the location is save, a reliability study of site candidate soil becomes very importance to be conducted through some experiments consisted some affected parameters such as contact time, effect of ionic strength, and effect of Sr{sup +} ion in solution. Radiostrontium was used as a tracer on the experiments and has role as radionuclide reference in low-level radioactive waste due to its long half-live and it's easy to associate with organism in nature. So, interaction of radiostrontium and soil samples from site becomes important to be studied. Experiment was performed in batch method, and soil sample-solution containing radionuclide was mixed in a 20 ml of PE vial. Ratio of solid: liquid was 10{sup −2} g/ml. Objective of the experiment is to collect the specific characteristics data of radionuclide sorption onto soil from site candidate. Distribution coefficient value was used as indicator where the amount of initial and final activities of radiostrontium in solution was compared. Result showed that equilibrium condition was reached after contact time 10 days with Kd values ranged from 1600-2350 ml/g. Increased in ionic strength in solution made decreased of Kd value into soil sample due to competition of background salt and radiostrontium into soil samples, and increased in Sr ion in solution caused decreased of Kd value in soil sample due to limitation of sorption capacity in soil samples. Fast condition in saturated of metal ion into soil samples was reached due to a simple reaction was occurred.

  16. Targeting Protein-Protein Interactions for Parasite Control

    PubMed Central

    Taylor, Christina M.; Fischer, Kerstin; Abubucker, Sahar; Wang, Zhengyuan; Martin, John; Jiang, Daojun; Magliano, Marc; Rosso, Marie-Noëlle; Li, Ben-Wen; Fischer, Peter U.; Mitreva, Makedonka

    2011-01-01

    Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific ortholgous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable. PMID

  17. Quantitative study of protein-protein interactions by quartz nanopipettes

    NASA Astrophysics Data System (ADS)

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-08-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with

  18. Prediction and redesign of protein-protein interactions.

    PubMed

    Lua, Rhonald C; Marciano, David C; Katsonis, Panagiotis; Adikesavan, Anbu K; Wilkins, Angela D; Lichtarge, Olivier

    2014-01-01

    Understanding the molecular basis of protein function remains a central goal of biology, with the hope to elucidate the role of human genes in health and in disease, and to rationally design therapies through targeted molecular perturbations. We review here some of the computational techniques and resources available for characterizing a critical aspect of protein function - those mediated by protein-protein interactions (PPI). We describe several applications and recent successes of the Evolutionary Trace (ET) in identifying molecular events and shapes that underlie protein function and specificity in both eukaryotes and prokaryotes. ET is a part of analytical approaches based on the successes and failures of evolution that enable the rational control of PPI. PMID:24878423

  19. The Arabidopsis ESCRT protein-protein interaction network.

    PubMed

    Shahriari, Mojgan; Richter, Klaus; Keshavaiah, Channa; Sabovljevic, Aneta; Huelskamp, Martin; Schellmann, Swen

    2011-05-01

    In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway. PMID:21442383

  20. Anisotropic interactions opposing magnetocrystalline anisotropy in Sr3NiIrO6

    NASA Astrophysics Data System (ADS)

    Lefrançois, E.; Pradipto, A.-M.; Moretti Sala, M.; Chapon, L. C.; Simonet, V.; Picozzi, S.; Lejay, P.; Petit, S.; Ballou, R.

    2016-06-01

    We report our investigation of the electronic and magnetic excitations of Sr3NiIrO6 by resonant inelastic x-ray scattering at the Ir L3 edge. The intra-t2 g electronic transitions are analyzed using an atomic model, including spin-orbit coupling and trigonal distortion of the IrO6 octahedron, confronted with ab initio quantum chemistry calculations. The Ir spin-orbital entanglement is quantified and its implications for the magnetic properties, in particular in inducing highly anisotropic magnetic interactions, are highlighted. These are included in the spin-wave model proposed to account for the dispersionless magnetic excitation that we observe at 90 m e V . By counterbalancing the strong Ni2 + easy-plane anisotropy that manifests itself at high temperature, the anisotropy of the interactions finally leads to the remarkable easy-axis magnetism reported in this material at low temperature.

  1. Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1

    PubMed Central

    Tang, Zhaohua; Kuo, Tiffany; Shen, Jenny; Lin, Ren-Jang

    2000-01-01

    Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore, dsk1+ gene determines the phenotype of prp2+ overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1+ and SRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms. PMID:10629038

  2. Self diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The

  3. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  4. Motif mediated protein-protein interactions as drug targets.

    PubMed

    Corbi-Verge, Carles; Kim, Philip M

    2016-01-01

    Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery. PMID:26936767

  5. Modulation of opioid receptor function by protein-protein interactions.

    PubMed

    Alfaras-Melainis, Konstantinos; Gomes, Ivone; Rozenfeld, Raphael; Zachariou, Venetia; Devi, Lakshmi

    2009-01-01

    Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified protein-protein interactions that play an essential role in opioid receptor function and responsiveness. Here, we summarize interactions shown to affect receptor biogenesis and trafficking, as well as those affecting signal transduction events following receptor activation. This article also examines protein interactions modulating the rate of receptor endocytosis and degradation, events that play a major role in opiate analgesia. Like several other GPCRs, opioid receptors may form homo or heterodimers. The last part of this review summarizes recent knowledge on proteins known to affect opioid receptor dimerization. PMID:19273296

  6. Docking and scoring protein interactions: CAPRI 2009.

    PubMed

    Lensink, Marc F; Wodak, Shoshana J

    2010-11-15

    Protein docking algorithms are assessed by evaluating blind predictions performed during 2007-2009 in Rounds 13-19 of the community-wide experiment on critical assessment of predicted interactions (CAPRI). We evaluated the ability of these algorithms to sample docking poses and to single out specific association modes in 14 targets, representing 11 distinct protein complexes. These complexes play important biological roles in RNA maturation, G-protein signal processing, and enzyme inhibition and function. One target involved protein-RNA interactions not previously considered in CAPRI, several others were hetero-oligomers, or featured multiple interfaces between the same protein pair. For most targets, predictions started from the experimentally determined structures of the free (unbound) components, or from models built from known structures of related or similar proteins. To succeed they therefore needed to account for conformational changes and model inaccuracies. In total, 64 groups and 12 web-servers submitted docking predictions of which 4420 were evaluated. Overall our assessment reveals that 67% of the groups, more than ever before, produced acceptable models or better for at least one target, with many groups submitting multiple high- and medium-accuracy models for two to six targets. Forty-one groups including four web-servers participated in the scoring experiment with 1296 evaluated models. Scoring predictions also show signs of progress evidenced from the large proportion of correct models submitted. But singling out the best models remains a challenge, which also adversely affects the ability to correctly rank docking models. With the increased interest in translating abstract protein interaction networks into realistic models of protein assemblies, the growing CAPRI community is actively developing more efficient and reliable docking and scoring methods for everyone to use. PMID:20806235

  7. Carbohydrate–Aromatic Interactions in Proteins

    PubMed Central

    2015-01-01

    Protein–carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C–H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C–H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C–H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate–aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C–H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein–carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein–carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites. PMID:26561965

  8. Male-specific phosphorylated SR proteins in adult flies of the Mediterranean Fruitfly Ceratitis capitata

    PubMed Central

    2014-01-01

    Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly. PMID:25472723

  9. Dynamic regulation of lipid-protein interactions.

    PubMed

    Martfeld, Ashley N; Rajagopalan, Venkatesan; Greathouse, Denise V; Koeppe, Roger E

    2015-09-01

    We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein-lipid interactions. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25666872

  10. Multifunctional proteins revealed by overlapping clustering in protein interaction network

    PubMed Central

    Chapple, Charles E.; Guénoche, Alain; Brun, Christine

    2012-01-01

    Motivation: Multifunctional proteins perform several functions. They are expected to interact specifically with distinct sets of partners, simultaneously or not, depending on the function performed. Current graph clustering methods usually allow a protein to belong to only one cluster, therefore impeding a realistic assignment of multifunctional proteins to clusters. Results: Here, we present Overlapping Cluster Generator (OCG), a novel clustering method which decomposes a network into overlapping clusters and which is, therefore, capable of correct assignment of multifunctional proteins. The principle of OCG is to cover the graph with initial overlapping classes that are iteratively fused into a hierarchy according to an extension of Newman's modularity function. By applying OCG to a human protein–protein interaction network, we show that multifunctional proteins are revealed at the intersection of clusters and demonstrate that the method outperforms other existing methods on simulated graphs and PPI networks. Availability: This software can be downloaded from http://tagc.univ-mrs.fr/welcome/spip.php?rubrique197 Contact: brun@tagc.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080466

  11. Dynamic network analysis of protein interactions

    NASA Astrophysics Data System (ADS)

    Almaas, Eivind; Deri, Joya

    2007-03-01

    Network approaches have recently become a popular tool to study complex systems such as cellular metabolism and protein interactions. A substantial number of analyses of the protein interaction network (PIN) of the yeast Saccharomyces cerevisiae have considered this network as a static entity, not taking the network's dynamic nature into account. Here, we examine the time-variation of gene regulation superimposed on the PIN by defining mRNA expression profiles throughout the cell cycle as node weights. To characterize these network dynamics, we have both developed a set of novel network measures as well as studied previously published measures for weighted networks. We expect that our approach will provide a deeper understanding of protein regulation during the cell cycle.

  12. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  13. A Tool for Interactive Protein Manipulation

    Energy Science and Technology Software Center (ESTSC)

    2005-03-28

    ProteinShop is a graphical environment that facilitates a solution to the protein prediction problem through a combination of unique features and capabilities. These include: 1. Helping researchers automatically generate 3D protein structures from scratcW by using the sequence of amino acids and secondary structure specifications as input. 2. Enabling users to apply their accumulated biochemical knowledge and intuition during the interactive manipulation of structures. 3. FacIlitating interactive comparison and analysis of alternative structures through visualizationmore » of free energy computed during modeling. 4. Accelerating discovery of low-energy configurations by applying local optimizations plug-in to user-selected protein structures. ProteinShop v.2.0 includes the following new features: - Visualizes multiple-domain structures - Automatically creates a user-specified number of beta-sheet configurations - Provides the interface and the libraries for energy visualization and local minimization of protein structures - Reads standard POB files without previous editing« less

  14. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  15. The centrality of cancer proteins in human protein-protein interaction network: a revisit.

    PubMed

    Xiong, Wei; Xie, Luyu; Zhou, Shuigeng; Liu, Hui; Guan, Jihong

    2014-01-01

    Topological analysis of protein-protein interaction (PPI) networks has been widely applied to the investigation on cancer mechanisms. However, there is still a debate on whether cancer proteins exhibit more topological centrality compared to the other proteins in the human PPI network. To resolve this debate, we first identified four sets of human proteins, and then mapped these proteins into the yeast PPI network by homologous genes. Finally, we compared these proteins' properties in human and yeast PPI networks. Experiments over two real datasets demonstrated that cancer proteins tend to have higher degree and smaller clustering coefficient than non-cancer proteins. Experimental results also validated that cancer proteins have larger betweenness centrality compared to the other proteins on the STRING dataset. However, on the BioGRID dataset, the average betweenness centrality of cancer proteins is larger than that of disease and control proteins, but smaller than that of essential proteins. PMID:24878726

  16. Tools for controlling protein interactions with light

    PubMed Central

    Tucker, Chandra L.; Vrana, Justin D.; Kennedy, Matthew J.

    2014-01-01

    Genetically-encoded actuators that allow control of protein-protein interactions with light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use controlling transcription, protein localization, and protein secretion with light. Additionally, we provide instructions and software for constructing a pulse-controlled LED light device for use in experiments requiring extended light treatments. PMID:25181301

  17. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  18. Predicting Disease-Related Proteins Based on Clique Backbone in Protein-Protein Interaction Network

    PubMed Central

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases. PMID:25013377

  19. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  20. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGESBeta

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  1. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  2. The Cellular EJC Interactome Reveals Higher Order mRNP Structure and an EJC-SR Protein Nexus

    PubMed Central

    Singh, Guramrit; Kucukural, Alper; Cenik, Can; Leszyk, John D.; Shaffer, Scott A.; Weng, Zhiping; Moore, Melissa J.

    2012-01-01

    SUMMARY In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction. PMID:23084401

  3. Ribo-Proteomics Approach to Profile RNA-Protein and Protein-Protein Interaction Networks.

    PubMed

    Yeh, Hsin-Sung; Chang, Jae-Woong; Yong, Jeongsik

    2016-01-01

    Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses. PMID:26965265

  4. Michigan molecular interactions r2: from interacting proteins to pathways.

    PubMed

    Tarcea, V Glenn; Weymouth, Terry; Ade, Alex; Bookvich, Aaron; Gao, Jing; Mahavisno, Vasudeva; Wright, Zach; Chapman, Adriane; Jayapandian, Magesh; Ozgür, Arzucan; Tian, Yuanyuan; Cavalcoli, Jim; Mirel, Barbara; Patel, Jignesh; Radev, Dragomir; Athey, Brian; States, David; Jagadish, H V

    2009-01-01

    Molecular interaction data exists in a number of repositories, each with its own data format, molecule identifier and information coverage. Michigan molecular interactions (MiMI) assists scientists searching through this profusion of molecular interaction data. The original release of MiMI gathered data from well-known protein interaction databases, and deep merged this information while keeping track of provenance. Based on the feedback received from users, MiMI has been completely redesigned. This article describes the resulting MiMI Release 2 (MiMIr2). New functionality includes extension from proteins to genes and to pathways; identification of highlighted sentences in source publications; seamless two-way linkage with Cytoscape; query facilities based on MeSH/GO terms and other concepts; approximate graph matching to find relevant pathways; support for querying in bulk; and a user focus-group driven interface design. MiMI is part of the NIH's; National Center for Integrative Biomedical Informatics (NCIBI) and is publicly available at: http://mimi.ncibi.org. PMID:18978014

  5. Protein-protein interactions of mitochondrial-associated protein via bioluminescence resonance energy transfer

    PubMed Central

    Koshiba, Takumi

    2015-01-01

    Protein-protein interactions are essential biological reactions occurring at inter- and intra-cellular levels. The analysis of their mechanism is generally required in order link to understand their various cellular functions. Bioluminescence resonance energy transfer (BRET), which is based on an enzymatic activity of luciferase, is a useful tool for investigating protein-protein interactions in live cells. The combination of the BRET system and biomolecular fluorescence complementation (BiFC) would provide us a better understanding of the hetero-oligomeric structural states of protein complexes. In this review, we discuss the application of BRET to the protein-protein interactions of mitochondrial-associated proteins and discuss its physiological relevance. PMID:27493852

  6. Turning the spotlight on protein-lipid interactions in cells

    PubMed Central

    Peng, Tao; Yuan, Xiaoqiu; Hang, Howard C.

    2014-01-01

    Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. While powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions. PMID:25129056

  7. Schizophrenia interactome with 504 novel protein-protein interactions.

    PubMed

    Ganapathiraju, Madhavi K; Thahir, Mohamed; Handen, Adam; Sarkar, Saumendra N; Sweet, Robert A; Nimgaonkar, Vishwajit L; Loscher, Christine E; Bauer, Eileen M; Chaparala, Srilakshmi

    2016-01-01

    Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein-protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities. PMID:27336055

  8. Interactions between Cs, Sr, and other nutrients and trace element accumulation in Amaranthus shoot in response to variety effect.

    PubMed

    Chu, Qingnan; Watanabe, Toshihiro; Sha, Zhimin; Osaki, Mitsuru; Shinano, Takuro

    2015-03-01

    Aiming at clarifying the interactions between Cs, Sr, and other mineral elements in the genus Amaranthus, this study adopted 33 different varieties of Amaranthus and investigated the concentrations of 23 mineral elements in shoots grown in the fields of Iino in Fukushima prefecture. Significant varietal effects were detected for all elements except Se, and degree of interspecies variation was highly element dependent. Among 23 elements, amaranths were less sensitive to the accumulation of Cs and Sr than most other mineral elements to the species level. There are six elements showing significant correlation with Cs, positive correlations between As, Rb, Al, Fe, Ni, and Cs, and negative correlation between Ba and Cs. Significant correlations between Ca, Mg, Mn, Zn, B, Ba, Cd, and Sr were detected, and all of the coefficients were positive. Cs and Sr did not present significant correlation, but they were both significantly correlated with Ba. By principal component analysis (PCA), the first and second principal components (PC1 and PC2) accounted for 23.2 and 20.3% of the total variance and associated with Cs and Sr, respectively. Both of the two species took up more Cs by promoting the influx of elements positively correlated with Cs into shoot, but at the same time, Amaranthus hypochondriacus (L.) Mapes 847 decreased the K and Ba uptake and Amaranthus powellii (S. Wats) subsp. Powellii inhibited the accumulation of Rb, Sr, and significantly correlated elements of Sr in shoot. This study is the first to pave the way for comprehension on ionome in amaranth shoot at the variety level. The results of this research provide the ionomic basis for implementing countermeasures in the field against the translocation of Cs (and potentially Sr) toward crops and food. PMID:25660261

  9. Notable Aspects of Glycan-Protein Interactions

    PubMed Central

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  10. Notable Aspects of Glycan-Protein Interactions.

    PubMed

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host's immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  11. Identification of essential proteins based on ranking edge-weights in protein-protein interaction networks.

    PubMed

    Wang, Yan; Sun, Huiyan; Du, Wei; Blanzieri, Enrico; Viero, Gabriella; Xu, Ying; Liang, Yanchun

    2014-01-01

    Essential proteins are those that are indispensable to cellular survival and development. Existing methods for essential protein identification generally rely on knock-out experiments and/or the relative density of their interactions (edges) with other proteins in a Protein-Protein Interaction (PPI) network. Here, we present a computational method, called EW, to first rank protein-protein interactions in terms of their Edge Weights, and then identify sub-PPI-networks consisting of only the highly-ranked edges and predict their proteins as essential proteins. We have applied this method to publicly-available PPI data on Saccharomyces cerevisiae (Yeast) and Escherichia coli (E. coli) for essential protein identification, and demonstrated that EW achieves better performance than the state-of-the-art methods in terms of the precision-recall and Jackknife measures. The highly-ranked protein-protein interactions by our prediction tend to be biologically significant in both the Yeast and E. coli PPI networks. Further analyses on systematically perturbed Yeast and E. coli PPI networks through randomly deleting edges demonstrate that the proposed method is robust and the top-ranked edges tend to be more associated with known essential proteins than the lowly-ranked edges. PMID:25268881

  12. Heparan sulfate and heparin interactions with proteins

    PubMed Central

    Meneghetti, Maria C. Z.; Hughes, Ashley J.; Rudd, Timothy R.; Nader, Helena B.; Powell, Andrew K.; Yates, Edwin A.; Lima, Marcelo A.

    2015-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed. PMID:26289657

  13. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

    PubMed Central

    Erkelenz, Steffen; Mueller, William F.; Evans, Melanie S.; Busch, Anke; Schöneweis, Katrin; Hertel, Klemens J.; Schaal, Heiner

    2013-01-01

    Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. PMID:23175589

  14. The influence of protein-protein interactions on the organization of proteins within thylakoid membranes.

    PubMed

    Tremmel, I G; Weis, E; Farquhar, G D

    2005-04-01

    The influence of attractive protein-protein interactions on the organization of photosynthetic proteins within the thylakoid membrane was investigated. Protein-protein interactions were simulated using Monte Carlo techniques and the influence of different interaction energies was examined. It was found that weak interactions led to protein clusters whereas strong interactions led to ramified chains. An optimum curve for the relationship between interaction energy and the number of contact sites emerged. With increasing particle densities the effect decreased. In a mixture of interacting and noninteracting particles the distance between the noninteracting particles was increased and there seemed to be much more free space around them. In thylakoids, this could lead to a more homogeneous distribution of the noninteracting but rate-limiting cytochrome bf complexes. Due to the increased free space between cytochrome bf, obstruction of binding sites--occurring unavoidably in a random distribution--may be drastically reduced. Furthermore, protein-protein interactions in thylakoids may lead to a decrease in plastoquinone diffusion. PMID:15665125

  15. Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

    PubMed Central

    Hastings, Michelle L.

    2014-01-01

    Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics. PMID:25506695

  16. Imaging Protein Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation

    PubMed Central

    Slavoff, Sarah A.; Liu, Daniel S.; Cohen, Justin D.; Ting, Alice Y.

    2012-01-01

    We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplAW37V, which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplAW37V labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein protein interactions can be imaged (FRB FKBP, Fos Jun, and neuroligin PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution. PMID:22098454

  17. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  18. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  19. Light-Scattering Studies of Protein Solutions: Role of Hydration in Weak Protein-Protein Interactions

    PubMed Central

    Paliwal, A.; Asthagiri, D.; Abras, D.; Lenhoff, A. M.; Paulaitis, M. E.

    2005-01-01

    We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B2, by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B2 and comparing our results with those derived from light-scattering measurements of B2 for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25°C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B2. We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration. PMID:15980182

  20. Algorithmic approaches to protein-protein interaction site prediction.

    PubMed

    Aumentado-Armstrong, Tristan T; Istrate, Bogdan; Murgita, Robert A

    2015-01-01

    Interaction sites on protein surfaces mediate virtually all biological activities, and their identification holds promise for disease treatment and drug design. Novel algorithmic approaches for the prediction of these sites have been produced at a rapid rate, and the field has seen significant advancement over the past decade. However, the most current methods have not yet been reviewed in a systematic and comprehensive fashion. Herein, we describe the intricacies of the biological theory, datasets, and features required for modern protein-protein interaction site (PPIS) prediction, and present an integrative analysis of the state-of-the-art algorithms and their performance. First, the major sources of data used by predictors are reviewed, including training sets, evaluation sets, and methods for their procurement. Then, the features employed and their importance in the biological characterization of PPISs are explored. This is followed by a discussion of the methodologies adopted in contemporary prediction programs, as well as their relative performance on the datasets most recently used for evaluation. In addition, the potential utility that PPIS identification holds for rational drug design, hotspot prediction, and computational molecular docking is described. Finally, an analysis of the most promising areas for future development of the field is presented. PMID:25713596

  1. Deciphering Supramolecular Structures with Protein-Protein Interaction Network Modeling

    PubMed Central

    Tsuji, Toshiyuki; Yoda, Takao; Shirai, Tsuyoshi

    2015-01-01

    Many biological molecules are assembled into supramolecules that are essential to perform complicated functions in the cell. However, experimental information about the structures of supramolecules is not sufficient at this point. We developed a method of predicting and modeling the structures of supramolecules in a biological network by combining structural data of the Protein Data Bank (PDB) and interaction data in IntAct databases. Templates for binary complexes in IntAct were extracted from PDB. Modeling was attempted by assembling binary complexes with superposed shared subunits. A total of 3,197 models were constructed, and 1,306 (41% of the total) contained at least one subunit absent from experimental structures. The models also suggested 970 (25% of the total) experimentally undetected subunit interfaces, and 41 human disease-related amino acid variants were mapped onto these model-suggested interfaces. The models demonstrated that protein-protein interaction network modeling is useful to fill the information gap between biological networks and structures. PMID:26549015

  2. Targeting protein-protein interactions as an anticancer strategy

    PubMed Central

    Ivanov, Andrei A.; Khuri, Fadlo R.; Fu, Haian

    2013-01-01

    The emergence and convergence of cancer genomics, targeted therapies, and network oncology have significantly expanded the landscape of protein-protein interaction (PPI) networks in cancer for therapeutic discovery. Extensive biological and clinical investigations have led to the identification of protein interaction hubs and nodes that are critical for the acquisition and maintaining characteristics of cancer essential for cell transformation. Such cancer enabling PPIs have become promising therapeutic targets. With technological advances in PPI modulator discovery and validation of PPI-targeting agents in clinical settings, targeting PPI interfaces as an anticancer strategy has become a reality. Future research directed at genomics-based PPI target discovery, PPI interface characterization, PPI-focused chemical library design, and patient-genomic subpopulation-driven clinical studies is expected to accelerate the development of the next generation of PPI-based anticancer agents for personalized precision medicine. Here we briefly review prominent PPIs that mediate cancer-acquired properties, highlight recognized challenges and promising clinical results in targeting PPIs, and outline emerging opportunities. PMID:23725674

  3. Parallel Force Assay for Protein-Protein Interactions

    PubMed Central

    Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

  4. Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum

    PubMed Central

    Rani, Shilpa; Park, Chang Sik; Sreenivasaiah, Pradeep Kumar; Kim, Do Han

    2016-01-01

    In the heart, excitation-contraction (E-C) coupling is mediated by Ca2+ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca2+ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca2+ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202–231). Second, in vitro binding assays were conducted to examine the Ca2+ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca2+ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca2+ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca2+ into SR at intermediate Ca2+ concentrations. PMID:26674963

  5. Module organization and variance in protein-protein interaction networks

    PubMed Central

    Lin, Chun-Yu; Lee, Tsai-Ling; Chiu, Yi-Yuan; Lin, Yi-Wei; Lo, Yu-Shu; Lin, Chih-Ta; Yang, Jinn-Moon

    2015-01-01

    A module is a group of closely related proteins that act in concert to perform specific biological functions through protein–protein interactions (PPIs) that occur in time and space. However, the underlying module organization and variance remain unclear. In this study, we collected module templates to infer respective module families, including 58,041 homologous modules in 1,678 species, and PPI families using searches of complete genomic database. We then derived PPI evolution scores and interface evolution scores to describe the module elements, including core and ring components. Functions of core components were highly correlated with those of essential genes. In comparison with ring components, core proteins/PPIs were conserved across multiple species. Subsequently, protein/module variance of PPI networks confirmed that core components form dynamic network hubs and play key roles in various biological functions. Based on the analyses of gene essentiality, module variance, and gene co-expression, we summarize the observations of module organization and variance as follows: 1) a module consists of core and ring components; 2) core components perform major biological functions and collaborate with ring components to execute certain functions in some cases; 3) core components are more conserved and essential during organizational changes in different biological states or conditions. PMID:25797237

  6. From Topology to Phenotype in Protein-Protein Interaction Networks

    NASA Astrophysics Data System (ADS)

    Pržulj, Nataša

    We have recently witnessed an explosion in biological network data along with the development of computational approaches for their analyses. This new interdisciplinary research area is an integral part of systems biology, promising to provide new insights into organizational principles of life, as well as into evolution and disease. However, there is a danger that the area might become hindered by several emerging issues. In particular, there is typically a weak link between biological and computational scientists, resulting in the use of simple computational techniques of limited potential to explain these complex biological data. Hence, there is a danger that the community might view the topological features of network data as mere statistics, ignoring the value of the information contained in these data. This might result in the imposition of scientific doctrines, such as scale-free-centric (on the modelling side) and genome-centric (on the biological side) opinions onto this nascent research area. In this chapter, we take a network science perspective and present a brief, high-level overview of the area, commenting on possible challenges ahead. We focus on protein-protein interaction networks (PINs) in which nodes correspond to proteins in a cell and edges to physical bindings between the proteins.

  7. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  8. Developing algorithms for predicting protein-protein interactions of homology modeled proteins.

    SciTech Connect

    Martin, Shawn Bryan; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Roe, Diana C.

    2006-01-01

    The goal of this project was to examine the protein-protein docking problem, especially as it relates to homology-based structures, identify the key bottlenecks in current software tools, and evaluate and prototype new algorithms that may be developed to improve these bottlenecks. This report describes the current challenges in the protein-protein docking problem: correctly predicting the binding site for the protein-protein interaction and correctly placing the sidechains. Two different and complementary approaches are taken that can help with the protein-protein docking problem. The first approach is to predict interaction sites prior to docking, and uses bioinformatics studies of protein-protein interactions to predict theses interaction site. The second approach is to improve validation of predicted complexes after docking, and uses an improved scoring function for evaluating proposed docked poses, incorporating a solvation term. This scoring function demonstrates significant improvement over current state-of-the art functions. Initial studies on both these approaches are promising, and argue for full development of these algorithms.

  9. Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

    PubMed

    Ouimet, Claire M; Shao, Hao; Rauch, Jennifer N; Dawod, Mohamed; Nordhues, Bryce; Dickey, Chad A; Gestwicki, Jason E; Kennedy, Robert T

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs. PMID:27434096

  10. Ortholog-based protein-protein interaction prediction and its application to inter-species interactions

    PubMed Central

    Lee, Sheng-An; Chan, Cheng-hsiung; Tsai, Chi-Hung; Lai, Jin-Mei; Wang, Feng-Sheng; Kao, Cheng-Yan; Huang, Chi-Ying F

    2008-01-01

    Background The rapid growth of protein-protein interaction (PPI) data has led to the emergence of PPI network analysis. Despite advances in high-throughput techniques, the interactomes of several model organisms are still far from complete. Therefore, it is desirable to expand these interactomes with ortholog-based and other methods. Results Orthologous pairs of 18 eukaryotic species were expanded and merged with experimental PPI datasets. The contributions of interologs from each species were evaluated. The expanded orthologous pairs enable the inference of interologs for various species. For example, more than 32,000 human interactions can be predicted. The same dataset has also been applied to the prediction of host-pathogen interactions. PPIs between P. falciparum calmodulin and several H. sapiens proteins are predicted, and these interactions may contribute to the maintenance of host cell Ca2+ concentration. Using comparisons with Bayesian and structure-based approaches, interactions between putative HSP40 homologs of P. falciparum and the H. sapiens TNF receptor associated factor family are revealed, suggesting a role for these interactions in the interference of the human immune response to P. falciparum. Conclusion The PPI datasets are available from POINT and POINeT . Further development of methods to predict host-pathogen interactions should incorporate multiple approaches in order to improve sensitivity, and should facilitate the identification of targets for drug discovery and design. PMID:19091010

  11. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  12. Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough

    PubMed Central

    Holmes, Rebecca K.; Tuck, Alex C.; Zhu, Chenchen; Dunn-Davies, Hywel R.; Kudla, Grzegorz; Clauder-Munster, Sandra; Granneman, Sander; Steinmetz, Lars M.; Guthrie, Christine; Tollervey, David

    2015-01-01

    Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3’ end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation. Tiling arrays and RNAPII mapping data revealed 3’ extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression. PMID:26694144

  13. Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough.

    PubMed

    Holmes, Rebecca K; Tuck, Alex C; Zhu, Chenchen; Dunn-Davies, Hywel R; Kudla, Grzegorz; Clauder-Munster, Sandra; Granneman, Sander; Steinmetz, Lars M; Guthrie, Christine; Tollervey, David

    2015-12-01

    Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression. PMID:26694144

  14. Fluorescence Studies of Protein Crystallization Interactions

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori; Forsythe, Elizabeth

    1999-01-01

    We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

  15. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  16. Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers. PMID:10022858

  17. Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells.

    PubMed

    Gao, Jie; Ma, Yue; Fu, Hua-Lin; Luo, Qian; Wang, Zhen; Xiao, Yu-Huan; Yang, Hao; Cui, Da-Xiang; Jin, Wei-Lin

    2016-05-01

    The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma. PMID:27113584

  18. Chromophore/protein and chromophore/anion interactions in halorhodopsin.

    PubMed Central

    Lanyi, J K; Zimányi, L; Nakanishi, K; Derguini, F; Okabe, M; Honig, B

    1988-01-01

    Halorhodopsin (HR), the light-driven chloride transport pigment of Halobacterium halobium, was bleached and reconstituted with retinal analogues with the pi electron system interrupted at different locations (dihydroretinals). The absorption maxima of the artificial pigments formed with the dihydroretinals are found to be very similar to those of the corresponding pigments formed by reconstitution of bacteriorhodopsin (BR) and sensory rhodopsin (SR). This strongly suggests that the distribution of charges around the retinal is similar in all three bacterial rhodopsins. Comparison of the primary, and proposed secondary, structures for HR and BR reveal conserved asparagine (asp) and arginine (arg) residues, which are likely candidates for the ionizable amino acids that interact with the retinal. In a second set of experiments absorption shifts due to the binding of anions to Sites I and II in HR, reconstituted with different retinal analogues, were used to estimate the locations of these binding sites relative to the retinal. Site I is localized near the Schiff base, and Site II near the ionone ring. On the basis of these results a structural model for HR is proposed, which accounts for the spectroscopic properties of HR in terms of the three buried arg residues and two of the buried asp residues in the protein. PMID:3345330

  19. Prediction of Protein-Protein Interaction Sites Based on Naive Bayes Classifier

    PubMed Central

    Geng, Haijiang; Lu, Tao; Lin, Xiao; Liu, Yu; Yan, Fangrong

    2015-01-01

    Protein functions through interactions with other proteins and biomolecules and these interactions occur on the so-called interface residues of the protein sequences. Identifying interface residues makes us better understand the biological mechanism of protein interaction. Meanwhile, information about the interface residues contributes to the understanding of metabolic, signal transduction networks and indicates directions in drug designing. In recent years, researchers have focused on developing new computational methods for predicting protein interface residues. Here we creatively used a 181-dimension protein sequence feature vector as input to the Naive Bayes Classifier- (NBC-) based method to predict interaction sites in protein-protein complexes interaction. The prediction of interaction sites in protein interactions is regarded as an amino acid residue binary classification problem by applying NBC with protein sequence features. Independent test results suggested that Naive Bayes Classifier-based method with the protein sequence features as input vectors performed well. PMID:26697220

  20. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  1. The Foundations of Protein-Ligand Interaction

    NASA Astrophysics Data System (ADS)

    Klebe, Gerhard

    For the specific design of a drug we must first answer the question: How does a drug achieve its activity? An active ingredient must, in order to develop its action, bind to a particular target molecule in the body. Usually this is a protein, but also nucleic acids in the form of RNA and DNA can be target structures for active agents. The most important condition for binding is at first that the active agent exhibits the correct size and shape in order to optimally fit into a cavity exposed to the surface of the protein, the "bindingpocket". It is further necessary for the surface properties of the ligand and protein to be mutually compatible to form specific interactions. In 1894 Emil Fischer compared the exact fit of a substrate for the catalytic centre of an enzyme with the picture of a "lock-and-key". Paul Ehrlich coined in 1913 "Corpora non agunt nisi fixata", literally "bodies do not work when they are not bound". He wanted to imply that active agents that are meant to kill bacteria or parasites must be "fixed" by them, i.e. linked to their structures. Both concepts form the starting point for any rational concept in the development of active pharmaceutical ingredients. In many respects they still apply today. A drug must, after being administered, reach its target and interact with a biological macromolecule. Specific agents have a large affinity and sufficient selectivity to bind to the macromolecule's active site. This is the only way they can develop the desired biological activity without side-effects.

  2. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  3. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  4. Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii.

    PubMed

    Bonente, Giulia; Ballottari, Matteo; Truong, Thuy B; Morosinotto, Tomas; Ahn, Tae K; Fleming, Graham R; Niyogi, Krishna K; Bassi, Roberto

    2011-01-01

    In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in

  5. Interplay between spin-orbit coupling and Hubbard interaction in SrIrO3 and related Pbnm perovskite oxides

    NASA Astrophysics Data System (ADS)

    Zeb, M. Ahsan; Kee, Hae-Young

    2012-08-01

    There has been a rapidly growing interest in the interplay between spin-orbit coupling (SOC) and the Hubbard interaction U in correlated materials. A current consensus is that the stronger the SOC, the smaller is the critical interaction Uc required for a spin-orbit Mott insulator, because the atomic SOC splits a band into different total angular momentum bands, narrowing the effective bandwidth. It was further claimed that at large enough SOC, the stronger the SOC, the weaker the Uc, because in general the effective SOC is enhanced with increasing electron-electron interaction strength. Contrary to this expectation, we find that, in orthorhombic perovskite oxides (Pbnm), the stronger the SOC, the bigger the Uc. This originates from a line of Dirac nodes in Jeff=1/2 bands near the Fermi level, inherited from a combination of the lattice structure and a large SOC. Due to this protected line of nodes, there are small hole and electron pockets in SrIrO3, and such a small density of states makes the Hubbard interaction less efficient in building a magnetic insulator. The full phase diagram in U vs SOC is obtained, where nonmagnetic semimetal, magnetic metal, and magnetic insulator are found. Magnetic ordering patterns beyond Uc are also presented. We further discuss implications of our finding in relation to other perovskites such as SrRhO3 and SrRuO3.

  6. Ultracold magnetically tunable interactions without radiative-charge-transfer losses between Ca+, Sr+, Ba+, and Yb+ ions and Cr atoms

    NASA Astrophysics Data System (ADS)

    Tomza, Michał

    2015-12-01

    The Ca+, Sr+, Ba+, and Yb+ ions immersed in an ultracold gas of the Cr atoms are proposed as experimentally feasible heteronuclear systems in which ion-atom interactions at ultralow temperatures can be controlled with magnetically tunable Feshbach resonances without charge transfer and radiative losses. Ab initio techniques are applied to investigate electronic-ground-state properties of the (CaCr)+, (SrCr)+, (BaCr)+, and (YbCr)+ molecular ions. The potential energy curves, permanent electric dipole moments, and static electric dipole polarizabilities are computed. The spin-restricted open-shell coupled-cluster method restricted to single, double, and noniterative triple excitations and the multireference configuration-interaction method restricted to single and double excitations are employed. The scalar relativistic effects are included within the small-core energy-consistent pseudopotentials. The leading long-range induction and dispersion interaction coefficients are also reported. Finally, magnetic Feshbach resonances between the Ca+, Sr+, Ba+, and Yb+ ions interacting with the Cr atoms are analyzed. The present proposal opens the way towards robust quantum simulations and computations with ultracold ion-atom systems free of radiative charge-transfer losses.

  7. Configuration interaction study on the ground and excited electronic states of the SrH molecule

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoting; Liang, Guiying; Zhang, Xiaomei; Xu, Haifeng; Yan, Bing

    2016-02-01

    High-level ab initio calculations on the ground and the excited states of the SrH molecule have been carried out utilizing the multi-reference configuration interaction method plus Davidson correction (MRCI+Q) method, with small-core relativistic effective core potentials together with the corresponding correlation consistent polarized valence basis sets. The potential energy curves (PECs) of the 16 Λ-S states have been obtained with the aid of the avoided crossing rule between electronic states of the same symmetry. The spectroscopic constants of the bound states were calculated, most of which have been reported for the first time, with those pertaining to the X2Σ+, A2П, B2Σ+, and A‧2Δ states being in line with the available experimental and theoretical values. The calculated spin-orbit matrix element indicates a strong interaction between the X2Σ+ and A2П states in the Franck-Condon region. The spin-orbit coupling (SOC) splits the lowest strongly bound X2Σ+, A2П, A‧2Δ, B2Σ+, and D2Σ+ states into 9 Ω states. For the D2Σ+ state, the SOC shifts the potential-well minimum to higher energy and shortens the bond length. The transition properties of the bound Λ-S states were predicated, including the transition dipole moments (TDMs), the Franck-Condon factors, and the radiative lifetimes. The lifetimes were calculated to be 34.2 ns (v‧=0) and 55.0 ns (v‧=0) for A2П and B2Σ+, in good agreement with the experimental results of 33.8±1.9 ns and 48.4±2.0 ns.

  8. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  9. Protein-protein interactions of PDE4 family members - Functions, interactions and therapeutic value.

    PubMed

    Klussmann, Enno

    2016-07-01

    The second messenger cyclic adenosine monophosphate (cAMP) is ubiquitous and directs a plethora of functions in all cells. Although theoretically freely diffusible through the cell from the site of its synthesis it is not evenly distributed. It rather is shaped into gradients and these gradients are established by phospodiesterases (PDEs), the only enzymes that hydrolyse cAMP and thereby terminate cAMP signalling upstream of cAMP's effector systems. Miles D. Houslay has devoted most of his scientific life highly successfully to a particular family of PDEs, the PDE4 family. The family is encoded by four genes and gives rise to around 20 enzymes, all with different functions. M. Houslay has discovered many of these functions and realised early on that PDE4 family enzymes are attractive drug targets in a variety of human diseases, but not their catalytic activity as that is encoded in conserved domains in all family members. He postulated that targeting the intracellular location would provide the specificity that modern innovative drugs require to improve disease conditions with fewer side effects than conventional drugs. Due to the wealth of M. Houslay's work, this article can only summarize some of his discoveries and, therefore, focuses on protein-protein interactions of PDE4. The aim is to discuss functions of selected protein-protein interactions and peptide spot technology, which M. Houslay introduced into the PDE4 field for identifying interacting domains. The therapeutic potential of PDE4 interactions will also be discussed. PMID:26498857

  10. Intricate protein-protein interactions in the cyanobacterial circadian clock.

    PubMed

    Egli, Martin

    2014-08-01

    The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism. PMID:24936066

  11. Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock*

    PubMed Central

    Egli, Martin

    2014-01-01

    The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism. PMID:24936066

  12. Specificity and non-specificity in RNA–protein interactions

    PubMed Central

    Jankowsky, Eckhard; Harris, Michael E.

    2016-01-01

    Gene expression is regulated by complex networks of interactions between RNAs and proteins. Proteins that interact with RNA have been traditionally viewed as either specific or non-specific; specific proteins interact preferentially with defined RNA sequence or structure motifs, whereas non-specific proteins interact with RNA sites devoid of such characteristics. Recent studies indicate that the binary “specific vs. non-specific” classification is insufficient to describe the full spectrum of RNA–protein interactions. Here, we review new methods that enable quantitative measurements of protein binding to large numbers of RNA variants, and the concepts aimed as describing resulting binding spectra: affinity distributions, comprehensive binding models and free energy landscapes. We discuss how these new methodologies and associated concepts enable work towards inclusive, quantitative models for specific and non-specific RNA–protein interactions. PMID:26285679

  13. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  14. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning. PMID:27400769

  15. Evolution of protein interactions: from interactomes to interfaces.

    PubMed

    Andreani, Jessica; Guerois, Raphael

    2014-07-15

    Protein-protein interactions lie at the heart of most cellular processes. Many experimental and computational studies aim to deepen our understanding of these interactions and improve our capacity to predict them. In this respect, the evolutionary perspective is most interesting, since the preservation of structure and function puts constraints on the evolution of proteins and their interactions. However, uncovering these constraints remains a challenge, and the description and detection of evolutionary signals in protein-protein interactions is currently a very active field of research. Here, we review recent works dissecting the mechanisms of protein-protein interaction evolution and exploring how to use evolutionary information to predict interactions, both at the global level of the interactome and at the detailed level of protein-protein interfaces. We first present to what extent protein-protein interactions are found to be conserved within interactomes and which properties can influence their conservation. We then discuss the evolutionary and co-evolutionary pressures applied on protein-protein interfaces. Finally, we describe how the computational prediction of interfaces can benefit from evolutionary inputs. PMID:24853495

  16. Role of Junctin Protein Interactions in Cellular Dynamics of Calsequestrin Polymer upon Calcium Perturbation*

    PubMed Central

    Lee, Keun Woo; Maeng, Jin-Soo; Choi, Jeong Yi; Lee, Yu Ran; Hwang, Chae Young; Park, Sung Sup; Park, Hyun Kyu; Chung, Bong Hyun; Lee, Seung-Goo; Kim, Yeon-Soo; Jeon, Hyesung; Eom, Soo Hyun; Kang, ChulHee; Kim, Do Han; Kwon, Ki-Sun

    2012-01-01

    Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca2+-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca2+ that were decondensed upon Ca2+ depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca2+ depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca2+ concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca2+ and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca2+ homeostasis. PMID:22123818

  17. Human protein S inhibits the uptake of AcLDL and expression of SR-A through Mer receptor tyrosine kinase in human macrophages

    PubMed Central

    Liao, Dan; Wang, Xinwen; Li, Min; Lin, Peter H.; Yao, Qizhi

    2009-01-01

    Human protein S is an anticoagulation protein. However, it is unknown whether protein S could regulate the expression and function of macrophage scavenger receptor A (SR-A) in macrophages. Human THP-1 monocytes and peripheral blood monocytes were differentiated into macrophages and then treated with physiological concentrations of human protein S. We found that protein S significantly reduced acetylated low-density lipoprotein (AcLDL) uptake and binding by macrophages and decreased the intracellular cholesteryl ester content. Protein S suppressed the expression of the SR-A at both mRNA and protein levels. Protein S reduced the SR-A promoter activity primarily through inhibition in the binding of transcription factors to the AP-1 promoter element in macrophages. Furthermore, human protein S could bind and induce phosphorylation of Mer receptor tyrosine kinase (Mer RTK). Soluble Mer protein or tyrosine kinase inhibitor herbimycin A effectively blocked the effects of protein S on AcLDL uptake. Immunohistochemical analysis revealed that the level of protein S was substantially increased in human atherosclerotic arteries. Thus, human protein S can inhibit the expression and activity of SR-A through Mer RTK in macrophages, suggesting that human protein S is a modulator for macrophage functions in uptaking of modified lipoproteins. PMID:18922854

  18. Probing calmodulin protein-protein interactions using high-content protein arrays.

    PubMed

    O'Connell, David J; Bauer, Mikael; Linse, Sara; Cahill, Dolores J

    2011-01-01

    The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular -Ca(2+)-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca(2+)-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile novel protein-protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell. PMID:21901608

  19. Collective prediction of protein functions from protein-protein interaction networks

    PubMed Central

    2014-01-01

    Background Automated assignment of functions to unknown proteins is one of the most important task in computational biology. The development of experimental methods for genome scale analysis of molecular interaction networks offers new ways to infer protein function from protein-protein interaction (PPI) network data. Existing techniques for collective classification (CC) usually increase accuracy for network data, wherein instances are interlinked with each other, using a large amount of labeled data for training. However, the labeled data are time-consuming and expensive to obtain. On the other hand, one can easily obtain large amount of unlabeled data. Thus, more sophisticated methods are needed to exploit the unlabeled data to increase prediction accuracy for protein function prediction. Results In this paper, we propose an effective Markov chain based CC algorithm (ICAM) to tackle the label deficiency problem in CC for interrelated proteins from PPI networks. Our idea is to model the problem using two distinct Markov chain classifiers to make separate predictions with regard to attribute features from protein data and relational features from relational information. The ICAM learning algorithm combines the results of the two classifiers to compute the ranks of labels to indicate the importance of a set of labels to an instance, and uses an ICA framework to iteratively refine the learning models for improving performance of protein function prediction from PPI networks in the paucity of labeled data. Conclusion Experimental results on the real-world Yeast protein-protein interaction datasets show that our proposed ICAM method is better than the other ICA-type methods given limited labeled training data. This approach can serve as a valuable tool for the study of protein function prediction from PPI networks. PMID:24564855

  20. Towards understanding pre-mRNA splicing mechanisms and the role of SR proteins.

    PubMed

    Sahebi, Mahbod; Hanafi, Mohamed M; van Wijnen, Andre J; Azizi, Parisa; Abiri, Rambod; Ashkani, Sadegh; Taheri, Sima

    2016-08-10

    Alternative pre-mRNA splicing provides a source of vast protein diversity by removing non-coding sequences (introns) and accurately linking different exonic regions in the correct reading frame. The regulation of alternative splicing is essential for various cellular functions in both pathological and physiological conditions. In eukaryotic cells, this process is commonly used to increase proteomic diversity and to control gene expression either co- or post-transcriptionally. Alternative splicing occurs within a megadalton-sized, multi-component machine consisting of RNA and proteins; during the splicing process, this complex undergoes dynamic changes via RNA-RNA, protein-protein and RNA-protein interactions. Co-transcriptional splicing functionally integrates the transcriptional machinery, thereby enabling the two processes to influence one another, whereas post-transcriptional splicing facilitates the coupling of RNA splicing with post-splicing events. This review addresses the structural aspects of spliceosomes and the mechanistic implications of their stepwise assembly on the regulation of pre-mRNA splicing. Moreover, the role of phosphorylation-based, signal-induced changes in the regulation of the splicing process is demonstrated. PMID:27154819

  1. Ensemble learning prediction of protein-protein interactions using proteins functional annotations.

    PubMed

    Saha, Indrajit; Zubek, Julian; Klingström, Tomas; Forsberg, Simon; Wikander, Johan; Kierczak, Marcin; Maulik, Ujjwal; Plewczynski, Dariusz

    2014-04-01

    Protein-protein interactions are important for the majority of biological processes. A significant number of computational methods have been developed to predict protein-protein interactions using protein sequence, structural and genomic data. Vast experimental data is publicly available on the Internet, but it is scattered across numerous databases. This fact motivated us to create and evaluate new high-throughput datasets of interacting proteins. We extracted interaction data from DIP, MINT, BioGRID and IntAct databases. Then we constructed descriptive features for machine learning purposes based on data from Gene Ontology and DOMINE. Thereafter, four well-established machine learning methods: Support Vector Machine, Random Forest, Decision Tree and Naïve Bayes, were used on these datasets to build an Ensemble Learning method based on majority voting. In cross-validation experiment, sensitivity exceeded 80% and classification/prediction accuracy reached 90% for the Ensemble Learning method. We extended the experiment to a bigger and more realistic dataset maintaining sensitivity over 70%. These results confirmed that our datasets are suitable for performing PPI prediction and Ensemble Learning method is well suited for this task. Both the processed PPI datasets and the software are available at . PMID:24469380

  2. Protein tertiary structure recognition using optimized Hamiltonians with local interactions.

    PubMed Central

    Goldstein, R A; Luthey-Schulten, Z A; Wolynes, P G

    1992-01-01

    Protein folding codes embodying local interactions including surface and secondary structure propensities and residue-residue contacts are optimized for a set of training proteins by using spin-glass theory. A screening method based on these codes correctly matches the structure of a set of test proteins with proteins of similar topology with 100% accuracy, even with limited sequence similarity between the test proteins and the structural homologs and the absence of any structurally similar proteins in the training set. PMID:1409599

  3. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Such networks are typically constructed using high throughput techniques (e.g., Yeast-2-Hybrid experiments). Of particular interest are hub proteins that can interact wit...

  4. A Laboratory-Intensive Course on the Experimental Study of Protein-Protein Interactions

    ERIC Educational Resources Information Center

    Witherow, D. Scott; Carson, Sue

    2011-01-01

    The study of protein-protein interactions is important to scientists in a wide range of disciplines. We present here the assessment of a lab-intensive course that teaches students techniques used to identify and further study protein-protein interactions. One of the unique elements of the course is that students perform a yeast two-hybrid screen…

  5. A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network

    PubMed Central

    Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia

    2014-01-01

    Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity. PMID:25405206

  6. Studying Protein-Protein Interactions in Budding Yeast Using Co-immunoprecipitation.

    PubMed

    Foltman, Magdalena; Sanchez-Diaz, Alberto

    2016-01-01

    Understanding protein-protein interactions and the architecture of protein complexes in which they work is essential to identify their biological role. Protein co-immunoprecipitation (co-IP) is an invaluable technique used in biochemistry allowing the identification of protein interactors. Here, we describe in detail an immunoaffinity purification protocol as a one-step or two-step immunoprecipitation from budding yeast Saccharomyces cerevisiae cells to subsequently detect interactions between proteins involved in the same biological process. PMID:26519317

  7. In Vivo Application of Photocleavable Protein Interaction Reporter Technology

    PubMed Central

    Yang, Li; Zheng, Chunxiang; Weisbrod, Chad R.; Tang, Xiaoting; Munske, Gerhard R.; Hoopmann, Michael R.; Eng, Jimmy K.; Bruce, James E.

    2012-01-01

    Summary In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to E. coli cells and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo inter-cross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology. PMID:22168182

  8. Interactions of proteins in gels, solutions and on surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Radha Perumal

    2006-12-01

    The study of protein interaction, identification and separation has applications in various fields relating to Biotechnology. In this research these aspects were investigated. The proteins albumin, casein, poly-L-lysine were studied. FITC and TRITC were used to fluorescently tag the proteins. Confocal microscopy was used to image the interaction of proteins. The migration of fluorescently tagged protein-salt aggregates on solid surfaces during electrophoresis was investigated using Confocal microscopy. The secondary structural modifications of proteins in solutions were investigated using FTIR micro spectroscopic imaging. The size of the colloids formed due to protein-protein interactions as a function of the protein concentrations were studied using DLS and their charges were found using zeta potential measurements. Based on DL.S and zeta potential measurements, a model is proposed for interactions of oppositely charged proteins. The nature of interaction was found using UV - Visual spectroscopy. It was found that oppositely charged proteins formed ionic bonds. It was also found that FITC molecule influenced the surface charge of albumin more than TRITC molecule. The effects of the influence of cell geometries upon Electro Osmotic Flow (EOF) were studied using neutrally charged fluorescent Polystyrene beads. Results showed that tagging proteins with fluorescent molecules influenced their mobility and interactions with other proteins. However no secondary structural modifications of the proteins were observed when oppositely charged proteins interacted. It was also observed that electrostatic interactions made oppositely charged proteins form large aggregates. The EOF was found to be dependent upon the ionic strength of the buffer, conductivity of the solid surfaces, distance from the surface and position of the electrodes in the electrophoretic cell.

  9. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. PMID:25640894

  10. Modularity in the evolution of yeast protein interaction network

    PubMed Central

    Ogishima, Soichi; Tanaka, Hiroshi; Nakaya, Jun

    2015-01-01

    Protein interaction networks are known to exhibit remarkable structures: scale-free and small-world and modular structures. To explain the evolutionary processes of protein interaction networks possessing scale-free and small-world structures, preferential attachment and duplication-divergence models have been proposed as mathematical models. Protein interaction networks are also known to exhibit another remarkable structural characteristic, modular structure. How the protein interaction networks became to exhibit modularity in their evolution? Here, we propose a hypothesis of modularity in the evolution of yeast protein interaction network based on molecular evolutionary evidence. We assigned yeast proteins into six evolutionary ages by constructing a phylogenetic profile. We found that all the almost half of hub proteins are evolutionarily new. Examining the evolutionary processes of protein complexes, functional modules and topological modules, we also found that member proteins of these modules tend to appear in one or two evolutionary ages. Moreover, proteins in protein complexes and topological modules show significantly low evolutionary rates than those not in these modules. Our results suggest a hypothesis of modularity in the evolution of yeast protein interaction network as systems evolution. PMID:25914446

  11. Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein*

    PubMed Central

    De Houwer, Stephanie; Demeulemeester, Jonas; Thys, Wannes; Taltynov, Oliver; Zmajkovicova, Katarina; Christ, Frauke; Debyser, Zeger

    2012-01-01

    Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP1, amino acids 170EHLKTAVQMAVFIHNFKRKGGI191) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids 214QKQITKIQNFRVYYR228 and 262RRKVKIIRDYGK273) that come together in the CTD tertiary structure to form an exposed antiparallel β-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication. PMID:22872638

  12. Extreme multifunctional proteins identified from a human protein interaction network

    PubMed Central

    Chapple, Charles E.; Robisson, Benoit; Spinelli, Lionel; Guien, Céline; Becker, Emmanuelle; Brun, Christine

    2015-01-01

    Moonlighting proteins are a subclass of multifunctional proteins whose functions are unrelated. Although they may play important roles in cells, there has been no large-scale method to identify them, nor any effort to characterize them as a group. Here, we propose the first method for the identification of ‘extreme multifunctional' proteins from an interactome as a first step to characterize moonlighting proteins. By combining network topological information with protein annotations, we identify 430 extreme multifunctional proteins (3% of the human interactome). We show that the candidates form a distinct sub-group of proteins, characterized by specific features, which form a signature of extreme multifunctionality. Overall, extreme multifunctional proteins are enriched in linear motifs and less intrinsically disordered than network hubs. We also provide MoonDB, a database containing information on all the candidates identified in the analysis and a set of manually curated human moonlighting proteins. PMID:26054620

  13. Course 1: Physics of Protein-DNA Interaction

    NASA Astrophysics Data System (ADS)

    Bruinsma, R. F.

    1 Introduction 1.1 The central dogma and bacterial gene expression 1.2 Molecular structure 2 Thermodynamics and kinetics of repressor-DNA interaction 2.1 Thermodynamics and the lac repressor 2.2 Kinetics of repressor-DNA interaction 3 DNA deformability and protein-DNA interaction 3.1 Introduction 3.2 The worm-like chain 3.3 The RST model 4 Electrostatics in water and protein-DNA interaction 4.1 Macro-ions and aqueous electrostatics 4.2 The primitive model 4.3 Manning condensation 4.4 Counter-ion release and non-specific protein-DNA interaction

  14. Folding superfunnel to describe cooperative folding of interacting proteins.

    PubMed

    Smeller, László

    2016-07-01

    This paper proposes a generalization of the well-known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein-protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein-protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009-1016. © 2016 Wiley Periodicals, Inc. PMID:27090200

  15. Globular and disordered—the non-identical twins in protein-protein interactions

    PubMed Central

    Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B.

    2015-01-01

    In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol−1. PMID:26217672

  16. Globular and disordered-the non-identical twins in protein-protein interactions.

    PubMed

    Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

    2015-01-01

    In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol(-1). PMID:26217672

  17. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  18. Membrane-Mediated Interaction between Strongly Anisotropic Protein Scaffolds

    PubMed Central

    Schweitzer, Yonatan; Kozlov, Michael M.

    2015-01-01

    Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains. PMID:25710602

  19. HKC: an algorithm to predict protein complexes in protein-protein interaction networks.

    PubMed

    Wang, Xiaomin; Wang, Zhengzhi; Ye, Jun

    2011-01-01

    With the availability of more and more genome-scale protein-protein interaction (PPI) networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to predict protein complexes by identifying overlapping clusters. The experiments on two data sets and two benchmarks show that our algorithm has relatively high F-measure and exhibits better performance compared with some other methods. PMID:22174556

  20. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  1. Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

    PubMed Central

    Walsh, Brian W.; Shapiro, Walker; Simmons, Lyle A.; Keck, James L.

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds. PMID:23536821

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

    PubMed

    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

  3. 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

  4. Design of Protein-Protein Interactions with a Novel Ensemble-Based Scoring Algorithm

    NASA Astrophysics Data System (ADS)

    Roberts, Kyle E.; Cushing, Patrick R.; Boisguerin, Prisca; Madden, Dean R.; Donald, Bruce R.

    Protein-protein interactions (PPIs) are vital for cell signaling, protein trafficking and localization, gene expression, and many other biological functions. Rational modification of PPI targets provides a mechanism to understand their function and importance. However, PPI systems often have many more degrees of freedom and flexibility than the small-molecule binding sites typically targeted by protein design algorithms. To handle these challenging design systems, we have built upon the computational protein design algorithm K * [8,19] to develop a new design algorithm to study protein-protein and protein-peptide interactions. We validated our algorithm through the design and experimental testing of novel peptide inhibitors.

  5. Visualization and targeted disruption of protein interactions in living cells.

    PubMed

    Herce, Henry D; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M Cristina

    2013-01-01

    Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species. PMID:24154492

  6. Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

    SciTech Connect

    Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M. )

    1991-05-01

    A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand binding studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.

  7. GWIDD: a comprehensive resource for genome-wide structural modeling of protein-protein interactions

    PubMed Central

    2012-01-01

    Protein-protein interactions are a key component of life processes. The knowledge of the three-dimensional structure of these interactions is important for understanding protein function. Genome-Wide Docking Database (http://gwidd.bioinformatics.ku.edu) offers an extensive source of data for structural studies of protein-protein complexes on genome scale. The current release of the database combines the available experimental data on the structure and characteristics of protein interactions with structural modeling of protein complexes for 771 organisms spanned over the entire universe of life from viruses to humans. The interactions are stored in a relational database with user-friendly interface that includes various search options. The search results can be interactively previewed; the structures, downloaded, along with the interaction characteristics. PMID:23245398

  8. PIMA: Protein-Protein interactions in Macromolecular Assembly - a web server for its Analysis and Visualization

    PubMed Central

    Kaleeckal Mathew, Oommen; Sowdhamini, Ramanathan

    2016-01-01

    Protein-protein interactions are essential for the basic biological machinery of the cell. This is important for processes like protein synthesis, enzyme kinetics, molecular assembly and signal transduction. A high number of macromolecular structural complexes are known due to recent advances in structure determination techniques. Therefore, it is of interest to develop an interactive tool to objectively analyze large protein complexes. Hence, we describe the development and utility of a web enabled application named ‘Protein-Protein Interaction in Macro-molecular Assembly’ (PIMA) for the analysis of large protein assemblies. The intricate details of physical interactions amongst protein subunits in a large complex are presented as simple user preferred interactive network diagrams PMID:27212837

  9. Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

    PubMed

    Busse, Ricarda A; Scacioc, Andreea; Schalk, Amanda M; Krick, Roswitha; Thumm, Michael; Kühnel, Karin

    2016-01-01

    Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method. PMID:26552682

  10. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Sembongi, H.; Litwin, T. R.; Gao, J.; Neuman, K. C.; Fearnley, I. M.; Spinazzola, A.; Walker, J. E.; Holt, I. J.

    2012-01-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion. PMID:22453275

  11. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms"). PMID:26871637

  12. Computational biology for target discovery and characterization: a feasibility study in protein-protein interaction detection

    SciTech Connect

    Zhou, C; Zemla, A

    2009-02-25

    In this work we developed new code for detecting putative multi-domain protein-protein interactions for a small network of bacterial pathogen proteins, and determined how structure-driven domain-fusion (DF) methods should be scaled up for whole-proteome analysis. Protein-protein interactions are of great interest in structural biology and are important for understanding the biology of pathogens. The ability to predict protein-protein interactions provides a means for development of anti-microbials that may interfer with key processes in pathogenicity. The function of a protein-protein complex can be elucidated through knowledge of its structure. The overall goal of this project was to determine the feasibility of extending current LLNL capabilities to produce a high-throughput systems bio-informatics capability for identification and characterization of putative interacting protein partners within known or suspected small protein networks. We extended an existing LLNL methodology for identification of putative protein-protein interacting partners (Chakicherla et al (in review)) by writing a new code to identify multi-domain-fusion linkages (3 or more per complex). We applied these codes to the proteins in the Yersinia pestis quorum sensing network, known as the lsr operon, which comprises a virulence mechanism in this pathogen. We determined that efficient application of our computational algorithms in high-throughput for detection of putative protein-protein complexes genome wide would require pre-computation of PDB domains and construction of a domain-domain association database.

  13. Structural study of surfactant-dependent interaction with protein

    SciTech Connect

    Mehan, Sumit; Aswal, Vinod K.; Kohlbrecher, Joachim

    2015-06-24

    Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

  14. PPLook: an automated data mining tool for protein-protein interaction

    PubMed Central

    2010-01-01

    Background Extracting and visualizing of protein-protein interaction (PPI) from text literatures are a meaningful topic in protein science. It assists the identification of interactions among proteins. There is a lack of tools to extract PPI, visualize and classify the results. Results We developed a PPI search system, termed PPLook, which automatically extracts and visualizes protein-protein interaction (PPI) from text. Given a query protein name, PPLook can search a dataset for other proteins interacting with it by using a keywords dictionary pattern-matching algorithm, and display the topological parameters, such as the number of nodes, edges, and connected components. The visualization component of PPLook enables us to view the interaction relationship among the proteins in a three-dimensional space based on the OpenGL graphics interface technology. PPLook can also provide the functions of selecting protein semantic class, counting the number of semantic class proteins which interact with query protein, counting the literature number of articles appearing the interaction relationship about the query protein. Moreover, PPLook provides heterogeneous search and a user-friendly graphical interface. Conclusions PPLook is an effective tool for biologists and biosystem developers who need to access PPI information from the literature. PPLook is freely available for non-commercial users at http://meta.usc.edu/softs/PPLook. PMID:20550717

  15. An ontology-based search engine for protein-protein interactions

    PubMed Central

    2010-01-01

    Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

  16. Electron-phonon interactions in superconducting La1.84Sr0.16CuO4 films.

    PubMed

    Shim, Heejae; Chaudhari, P; Logvenov, Gennady; Bozovic, Ivan

    2008-12-12

    We have measured quasiparticle tunneling across a junction perpendicular to the superconducting copper oxide planes. The tunneling spectra show peaks in the density of states. There are 11 minima in the second derivative d2I/dV2, where I is the current and V the voltage, suggesting multiple boson-quasiparticle interactions. These minima match precisely with the published Raman scattering data, leading us to conclude that the relevant bosons in superconducting La1.84Sr0.16CuO4 films are phonons. PMID:19113657

  17. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes. PMID:16901108

  18. Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins.

    PubMed

    Wang, Li; Xu, Yuan; Zhou, Sumei; Qian, Haifeng; Zhang, Hui; Qi, Xiguang; Fan, Meihua

    2016-03-01

    In this study, we investigated the interaction of Vaccinium bracteatum Thunb. leaf (VBTL) pigment and rice proteins. In the presence of rice protein, VBTL pigment antioxidant activity and free polyphenol content decreased by 67.19% and 68.11%, respectively, and L(∗) of the protein-pigment complex decreased significantly over time. L(∗) values of albumin, globulin and glutelin during 60-min pigment exposure decreased by 55.00, 57.14, and 54.30%, respectively, indicating that these proteins had bound to the pigment. A significant difference in protein surface hydrophobicity was observed between rice proteins and pigment-protein complexes, indicating that hydrophobic interaction is a major binding mechanism between VBTL pigment and rice proteins. A significant difference in secondary structures between proteins and protein-pigment complexes was also uncovered, indicating that hydrogen bonding may be another mode of interaction between VBTL pigment and rice proteins. Our results indicate that VBTL pigment can stain rice proteins with hydrophobic and hydrogen interactions. PMID:26471554

  19. Making the LINC: SUN and KASH protein interactions

    PubMed Central

    Kim, Dae In; Birendra, KC; Roux, Kyle J.

    2015-01-01

    Cell nuclei are physically integrated with the cytoskeleton through the LINC complex (for LInker of Nucleoskeleton and Cytoskeleton), a structure that spans the nuclear envelope to link the nucleoskeleton and cytoskeleton. Outer nuclear membrane KASH domain proteins and inner nuclear membrane SUN domain proteins interact to form the core of the LINC complex. In this review we provide a comprehensive analysis of the reported protein-protein interactions for KASH and SUN domain proteins. This critical structure, directly connecting the genome with the rest of the cell, contributes to a myriad of cellular functions and, when perturbed, is associated with human disease. PMID:25720065

  20. Interacting proteins as genetic modifiers of Huntington disease.

    PubMed

    Li, Xiao-Jiang; Friedman, Meyer; Li, Shihua

    2007-11-01

    Huntington disease is caused by polyglutamine expansion in huntingtin, a 350 kD protein that is ubiquitously expressed and widely distributed at the subcellular level. Recently, Kaltenbach et al. identified a large collection of novel huntingtin-interacting proteins, several of which modify mutant huntingtin toxicity in Drosophila. Thus, the interaction of mutant huntingtin with certain protein partners can influence its toxicity and therefore the severity and/or progression of Huntington disease. PMID:17961788

  1. Protein lethality investigated in terms of long range dynamical interactions.

    PubMed

    Rodrigues, Francisco A; Costa, Luciano da Fontoura

    2009-04-01

    The relationship between network structure/dynamics and biological function constitutes a fundamental issue in systems biology. However, despite many related investigations, the correspondence between structure and biological functions is not yet fully understood. A related subject that has deserved particular attention recently concerns how essentiality is related to the structure and dynamics of protein interactions. In the current work, protein essentiality is investigated in terms of long range influences in protein-protein interaction networks by considering simulated dynamical aspects. This analysis is performed with respect to outward activations, an approach which models the propagation of interactions between proteins by considering self-avoiding random walks. The obtained results are compared to protein local connectivity. Both the connectivity and the outward activations were found to be strongly related to protein essentiality. PMID:19396375

  2. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

    2002-10-15

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  3. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    PubMed

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species. PMID:25787689

  4. Structural Similarity and Classification of Protein Interaction Interfaces

    PubMed Central

    Zhao, Nan; Pang, Bin; Shyu, Chi-Ren; Korkin, Dmitry

    2011-01-01

    Interactions between proteins play a key role in many cellular processes. Studying protein-protein interactions that share similar interaction interfaces may shed light on their evolution and could be helpful in elucidating the mechanisms behind stability and dynamics of the protein complexes. When two complexes share structurally similar subunits, the similarity of the interaction interfaces can be found through a structural superposition of the subunits. However, an accurate detection of similarity between the protein complexes containing subunits of unrelated structure remains an open problem. Here, we present an alignment-free machine learning approach to measure interface similarity. The approach relies on the feature-based representation of protein interfaces and does not depend on the superposition of the interacting subunit pairs. Specifically, we develop an SVM classifier of similar and dissimilar interfaces and derive a feature-based interface similarity measure. Next, the similarity measure is applied to a set of 2,806×2,806 binary complex pairs to build a hierarchical classification of protein-protein interactions. Finally, we explore case studies of similar interfaces from each level of the hierarchy, considering cases when the subunits forming interactions are either homologous or structurally unrelated. The analysis has suggested that the positions of charged residues in the homologous interfaces are not necessarily conserved and may exhibit more complex conservation patterns. PMID:21589874

  5. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  6. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  7. The interaction of IQGAPs with calmodulin-like proteins.

    PubMed

    Pathmanathan, Sevvel; Hamilton, Elaine; Atcheson, Erwan; Timson, David J

    2011-04-01

    Since their identification over 15 years ago, the IQGAP (IQ-motif-containing GTPase-activating protein) family of proteins have been implicated in a wide range of cellular processes, including cytoskeletal reorganization, cell-cell adhesion, cytokinesis and apoptosis. These processes rely on protein-protein interactions, and understanding these (and how they influence one another) is critical in determining how the IQGAPs function. A key group of interactions is with calmodulin and the structurally related proteins myosin essential light chain and S100B. These interactions occur primarily through a series of IQ motifs, which are α-helical segments of the protein located towards the middle of the primary sequence. The three human IQGAP isoforms (IQGAP1, IQGAP2 and IQGAP3) all have four IQ motifs. However, these have different affinities for calmodulin, myosin light chain and S100B. Whereas all four IQ motifs of IQGAP1 interact with calmodulin in the presence of calcium, only the last two do so in the absence of calcium. IQ1 (the first IQ motif) interacts with the myosin essential light chain Mlc1sa and the first two undergo a calcium-dependent interaction with S100B. The significance of the interaction between Mlc1sa and IQGAP1 in mammals is unknown. However, a similar interaction involving the Saccharomyces cerevisiae IQGAP-like protein Iqg1p is involved in cytokinesis, leading to speculation that there may be a similar role in mammals. PMID:21428964

  8. Information-driven structural modelling of protein-protein interactions.

    PubMed

    Rodrigues, João P G L M; Karaca, Ezgi; Bonvin, Alexandre M J J

    2015-01-01

    Protein-protein docking aims at predicting the three-dimensional structure of a protein complex starting from the free forms of the individual partners. As assessed in the CAPRI community-wide experiment, the most successful docking algorithms combine pure laws of physics with information derived from various experimental or bioinformatics sources. Of these so-called "information-driven" approaches, HADDOCK stands out as one of the most successful representatives. In this chapter, we briefly summarize which experimental information can be used to drive the docking prediction in HADDOCK, and then focus on the docking protocol itself. We discuss and illustrate with a tutorial example a "classical" protein-protein docking prediction, as well as more recent developments for modelling multi-body systems and large conformational changes. PMID:25330973

  9. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  10. Restraint of the G2/M transition by the SR/RRM family mRNA shuttling binding protein SNXAHRB1 in Aspergillus nidulans.

    PubMed

    James, Steven W; Banta, Travis; Barra, James; Ciraku, Lorela; Coile, Clifford; Cuda, Zach; Day, Ryan; Dixit, Cheshil; Eastlack, Steven; Giang, Anh; Goode, James; Guice, Alexis; Huff, Yulon; Humbert, Sara; Kelliher, Christina; Kobie, Julie; Kohlbrenner, Emily; Mwambutsa, Faustin; Orzechowski, Amanda; Shingler, Kristin; Spell, Casey; Anglin, Sarah Lea

    2014-10-01

    Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2(cdc2) that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2(cdc) (2), nimE6(cyclinB), and nimT23(cdc) (25), but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10(cyclinB) mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA(Hrb) (1) is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA(HRB1) is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2(CDC2)/NIME(CYCLINB) kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIME(CYCLINB) at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. PMID:25104516

  11. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

    PubMed

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping

  12. Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

    PubMed Central

    Estruch, Sara B.; Fisher, Simon E.

    2014-01-01

    Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA. PMID:24893771

  13. The variable C-terminus of cysteine string proteins modulates exocytosis and protein-protein interactions.

    PubMed

    Boal, Frédéric; Zhang, Hui; Tessier, Céline; Scotti, Pier; Lang, Jochen

    2004-12-28

    Cysteine string proteins (Csps) are vesicle proteins involved in neurotransmission and hormone exocytosis. They are composed of distinct domains: a variable N-terminus, a J-domain followed by a linker region, a cysteine-rich string, and a C-terminus which diverges among isoforms. Their precise function and interactions are not fully understood. Using insulin exocytosis as a model, we show that the linker region and the C-terminus, but not the variable N-terminus, regulate overall secretion. Moreover, endogenous Csp1 binds in a calcium-dependent manner to monomeric VAMP2, and this interaction requires the C-terminus of Csp. The interaction is isoform specific as recombinant Csp1 binds VAMP1 and VAMP7, but not VAMP3. Cross-linking in permeabilized clonal beta-cells revealed homodimerization of Csp which is stimulated by Ca(2+) and again modulated by the variant C-terminus. Our data suggest that both interactions of Csp occur during exocytosis and may explain the effect of the variant C-terminus of this chaperon protein on peptide hormone secretion. PMID:15610015

  14. A Microfluidic Platform for Characterization of Protein—Protein Interactions

    PubMed Central

    Javanmard, Mehdi; Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Huber, David E.; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W.

    2010-01-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein–protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein–glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  15. Predicting Pharmacodynamic Drug-Drug Interactions through Signaling Propagation Interference on Protein-Protein Interaction Networks

    PubMed Central

    Park, Kyunghyun; Kim, Docyong; Ha, Suhyun; Lee, Doheon

    2015-01-01

    As pharmacodynamic drug-drug interactions (PD DDIs) could lead to severe adverse effects in patients, it is important to identify potential PD DDIs in drug development. The signaling starting from drug targets is propagated through protein-protein interaction (PPI) networks. PD DDIs could occur by close interference on the same targets or within the same pathways as well as distant interference through cross-talking pathways. However, most of the previous approaches have considered only close interference by measuring distances between drug targets or comparing target neighbors. We have applied a random walk with restart algorithm to simulate signaling propagation from drug targets in order to capture the possibility of their distant interference. Cross validation with DrugBank and Kyoto Encyclopedia of Genes and Genomes DRUG shows that the proposed method outperforms the previous methods significantly. We also provide a web service with which PD DDIs for drug pairs can be analyzed at http://biosoft.kaist.ac.kr/targetrw. PMID:26469276

  16. Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

    PubMed Central

    Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

    2016-01-01

    To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

  17. Probing Protein-DNA Interactions by Unzipping DNA

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2003-03-01

    Protein-DNA interactions are essential to cellular processes. In replication, transcription, recombination, DNA repair, and DNA packaging, proteins bind to DNA as activators or repressors, to recruit other proteins, or to carry out various catalytic activities. I will present Unzipping Force Analysis of Protein Association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule, we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex.

  18. The interaction of Ag with Bi-Pb-Sr-Ca-Cu-O superconductor

    NASA Astrophysics Data System (ADS)

    Dou, S. X.; Song, K. H.; Liu, H. K.; Sorrell, C. C.; Apperley, M. H.; Gouch, A. J.; Savvides, N.; Hensley, D. W.

    1989-10-01

    Bi-Pb-Sr-Ca-Cu-O superconductor compounds have been doped with up to 30 wt% Ag, sintered under variable oxygen partial pressure, and characterised in terms of the electrical and crystallographic behaviour. In contrast to previous reports that claim that Ag is the only metal non-poisoning to the superconductivity of Bi-Sr-Ca-Cu-O (BSCCO), it has been found that Ag additions to Bi-Pb-Sr-Ca-Cu-O depress Tc and Jc drastically and cause a large decrease in lattice parameters when samples are treated in air or pure oxygen. However, the lattice parameters, Tc and Jc remain unaffected by Ag additions when samples are heat treated in 0.030-0.067 atm oxygen. It is clear that the Ag reacts with and destabilises the superconducting phase when the samples are treated in air or pure oxygen while, when the samples are heat treated in low oxygen partial pressures, the Ag remains as an isolated inert metal phase that improves the weak links between the grains. This discovery clearly shows the feasibility of Ag-clad superconductor wire. For Ag-clad superconductor tape of 0.1 mm 2 cross sectional area heat treated in air, Jc was measured to be 54 A/cm 2. The same specimen sintered in 0.067 atm oxygen showed that the Jc increased to 2078 A/cm 2.

  19. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  20. A novel method for protein-protein interaction site prediction using phylogenetic substitution models

    PubMed Central

    La, David; Kihara, Daisuke

    2011-01-01

    Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. Based on this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and non-binding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins. PMID:21989996

  1. Network representation of protein interactions-Experimental results.

    PubMed

    Kurzbach, Dennis; Flamm, Andrea G; Sára, Tomáš

    2016-09-01

    A graph theoretical analysis of nuclear magnetic resonance (NMR) data of six different protein interactions has been presented. The representation of the protein interaction data as a graph or network reveals that all of the studied interactions are based on a common functional concept. They all involve a single densely packed hub of functionally correlated residues that mediate the ligand binding events. This is found independent of the kind of protein (folded or unfolded) or ligand (protein, polymer or small molecule). Furthermore, the power of the graph analysis is demonstrated at the examples of the Calmodulin (CaM)/Calcium and the Cold Shock Protein A (CspA)/RNA interaction. The presented approach enables the precise determination of multiple binding sites for the respective ligand molecules. PMID:27272395

  2. Single Molecule Approaches in RNA-Protein Interactions.

    PubMed

    Serebrov, Victor; Moore, Melissa J

    2016-01-01

    RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques. PMID:27256383

  3. [Multiparticle computer simulation of protein interactions in the photosynthetic membrane].

    PubMed

    Riznichenko, G Iu; Kovalenko, I B; Abaturova, A M; D'iakonova, A N; Kniazeva, O S; Ustinin, D M; Khrushchev, S S; Rubin, A B

    2011-01-01

    The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the protein plastocyanin with the protein cytochrome f and the pigment-protein complex of photosystem I, as well as of the protein ferredoxin with the protein FNR and photosystem 1 are considered. The role of diffusion and electrostatic interactions is discussed, and the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed. PMID:22117434

  4. Domain-mediated protein interaction prediction: From genome to network.

    PubMed

    Reimand, Jüri; Hui, Shirley; Jain, Shobhit; Law, Brian; Bader, Gary D

    2012-08-14

    Protein-protein interactions (PPIs), involved in many biological processes such as cellular signaling, are ultimately encoded in the genome. Solving the problem of predicting protein interactions from the genome sequence will lead to increased understanding of complex networks, evolution and human disease. We can learn the relationship between genomes and networks by focusing on an easily approachable subset of high-resolution protein interactions that are mediated by peptide recognition modules (PRMs) such as PDZ, WW and SH3 domains. This review focuses on computational prediction and analysis of PRM-mediated networks and discusses sequence- and structure-based interaction predictors, techniques and datasets for identifying physiologically relevant PPIs, and interpreting high-resolution interaction networks in the context of evolution and human disease. PMID:22561014

  5. Aromatic-aromatic interactions: analysis of π-π interactions in interleukins and TNF proteins

    PubMed Central

    Sivasakthi, Vaideeswaran; Anitha, Parimelzaghan; Kumar, Kalavathi Murugan; Bag, Susmita; Senthilvel, Padmanaban; Lavanya, Pandian; Swetha, Rayapadi; Anbarasu, Anand; Ramaiah, Sudha

    2013-01-01

    Aromatic-aromatic hydrogen bonds are important in many areas of chemistry, biology and materials science. In this study we have analyzed the roles played by the π-π interactions in interleukins (ILs) and tumor necrosis factor (TNF) proteins. Majority of π-π interacting residues are conserved in ILs and TNF proteins. The accessible surface area calculations in these proteins reveal that these interactions might be important in stabilizing the inner core regions of these proteins. In addition to π-π interactions, the aromatic residues also form π-networks in ILs and TNF proteins. The results obtained in the present study indicate that π-π interactions and π-π networks play important roles in the structural stability of ILs and TNF proteins. PMID:23750094

  6. Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

    PubMed

    Pujhari, Sujit; Zakhartchouk, Alexander N

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase. We additionally demonstrate partial mitochondrial localization of the E protein in transfected cells. To functionally investigate these interactions, we infected MARC-145 cells with PRRSV or alphavirus replicon particles (VRPs) expressing PRRSV E protein. In infected cells, production of ATP was significantly reduced. The E protein also induced apoptosis by activating caspase-3, which results in PARP cleavage. Taken together, these data suggest that the PRRSV E protein interacts with mitochondrial proteins and induces apoptosis by inhibiting ATP production. PMID:27068165

  7. Fermi Surface of Sr_{2}RuO_{4}: Spin-Orbit and Anisotropic Coulomb Interaction Effects.

    PubMed

    Zhang, Guoren; Gorelov, Evgeny; Sarvestani, Esmaeel; Pavarini, Eva

    2016-03-11

    The topology of the Fermi surface of Sr_{2}RuO_{4} is well described by local-density approximation calculations with spin-orbit interaction, but the relative size of its different sheets is not. By accounting for many-body effects via dynamical mean-field theory, we show that the standard isotropic Coulomb interaction alone worsens or does not correct this discrepancy. In order to reproduce experiments, it is essential to account for the Coulomb anisotropy. The latter is small but has strong effects; it competes with the Coulomb-enhanced spin-orbit coupling and the isotropic Coulomb term in determining the Fermi surface shape. Its effects are likely sizable in other correlated multiorbital systems. In addition, we find that the low-energy self-energy matrix-responsible for the reshaping of the Fermi surface-sizably differs from the static Hartree-Fock limit. Finally, we find a strong spin-orbital entanglement; this supports the view that the conventional description of Cooper pairs via factorized spin and orbital part might not apply to Sr_{2}RuO_{4}. PMID:27015496

  8. A proteome-wide protein interaction map for Campylobacter jejuni

    PubMed Central

    Parrish, Jodi R; Yu, Jingkai; Liu, Guozhen; Hines, Julie A; Chan, Jason E; Mangiola, Bernie A; Zhang, Huamei; Pacifico, Svetlana; Fotouhi, Farshad; DiRita, Victor J; Ideker, Trey; Andrews, Phillip; Finley, Russell L

    2007-01-01

    Background Data from large-scale protein interaction screens for humans and model eukaryotes have been invaluable for developing systems-level models of biological processes. Despite this value, only a limited amount of interaction data is available for prokaryotes. Here we report the systematic identification of protein interactions for the bacterium Campylobacter jejuni, a food-borne pathogen and a major cause of gastroenteritis worldwide. Results Using high-throughput yeast two-hybrid screens we detected and reproduced 11,687 interactions. The resulting interaction map includes 80% of the predicted C. jejuni NCTC11168 proteins and places a large number of poorly characterized proteins into networks that provide initial clues about their functions. We used the map to identify a number of conserved subnetworks by comparison to protein networks from Escherichia coli and Saccharomyces cerevisiae. We also demonstrate the value of the interactome data for mapping biological pathways by identifying the C. jejuni chemotaxis pathway. Finally, the interaction map also includes a large subnetwork of putative essential genes that may be used to identify potential new antimicrobial drug targets for C. jejuni and related organisms. Conclusion The C. jejuni protein interaction map is one of the most comprehensive yet determined for a free-living organism and nearly doubles the binary interactions available for the prokaryotic kingdom. This high level of coverage facilitates pathway mapping and function prediction for a large number of C. jejuni proteins as well as orthologous proteins from other organisms. The broad coverage also facilitates cross-species comparisons for the identification of evolutionarily conserved subnetworks of protein interactions. PMID:17615063

  9. Restraint of the G2/M Transition by the SR/RRM Family mRNA Shuttling Binding Protein SNXAHRB1 in Aspergillus nidulans

    PubMed Central

    James, Steven W.; Banta, Travis; Barra, James; Ciraku, Lorela; Coile, Clifford; Cuda, Zach; Day, Ryan; Dixit, Cheshil; Eastlack, Steven; Giang, Anh; Goode, James; Guice, Alexis; Huff, Yulon; Humbert, Sara; Kelliher, Christina; Kobie, Julie; Kohlbrenner, Emily; Mwambutsa, Faustin; Orzechowski, Amanda; Shingler, Kristin; Spell, Casey; Anglin, Sarah Lea

    2014-01-01

    Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein–protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2cdc2, nimE6cyclinB, and nimT23cdc25, but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxAHrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXAHRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2CDC2/NIMECYCLINB kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIMECYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. PMID:25104516

  10. Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

    PubMed Central

    DeBlasio, Stacy L.; Chavez, Juan D.; Alexander, Mariko M.; Ramsey, John; Eng, Jimmy K.; Mahoney, Jaclyn; Gray, Stewart M.; Bruce, James E.

    2015-01-01

    ABSTRACT Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection—hallmarks of host-pathogen interactions—were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCE The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used

  11. Discovering novel protein-protein interactions by measuring the protein semantic similarity from the biomedical literature.

    PubMed

    Chiang, Jung-Hsien; Ju, Jiun-Huang

    2014-12-01

    Protein-protein interactions (PPIs) are involved in the majority of biological processes. Identification of PPIs is therefore one of the key aims of biological research. Although there are many databases of PPIs, many other unidentified PPIs could be buried in the biomedical literature. Therefore, automated identification of PPIs from biomedical literature repositories could be used to discover otherwise hidden interactions. Search engines, such as Google, have been successfully applied to measure the relatedness among words. Inspired by such approaches, we propose a novel method to identify PPIs through semantic similarity measures among protein mentions. We define six semantic similarity measures as features based on the page counts retrieved from the MEDLINE database. A machine learning classifier, Random Forest, is trained using the above features. The proposed approach achieve an averaged micro-F of 71.28% and an averaged macro-F of 64.03% over five PPI corpora, an improvement over the results of using only the conventional co-occurrence feature (averaged micro-F of 68.79% and an averaged macro-F of 60.49%). A relation-word reinforcement further improves the averaged micro-F to 71.3% and averaged macro-F to 65.12%. Comparing the results of the current work with other studies on the AIMed corpus (ranging from 77.58% to 85.1% in micro-F, 62.18% to 76.27% in macro-F), we show that the proposed approach achieves micro-F of 81.88% and macro-F of 64.01% without the use of sophisticated feature extraction. Finally, we manually examine the newly discovered PPI pairs based on a literature review, and the results suggest that our approach could extract novel protein-protein interactions. PMID:25385082

  12. The origins of the evolutionary signal used to predict protein-protein interactions

    PubMed Central

    2012-01-01

    Background The correlation of genetic distances between pairs of protein sequence alignments has been used to infer protein-protein interactions. It has been suggested that these correlations are based on the signal of co-evolution between interacting proteins. However, although mutations in different proteins associated with maintaining an interaction clearly occur (particularly in binding interfaces and neighbourhoods), many other factors contribute to correlated rates of sequence evolution. Proteins in the same genome are usually linked by shared evolutionary history and so it would be expected that there would be topological similarities in their phylogenetic trees, whether they are interacting or not. For this reason the underlying species tree is often corrected for. Moreover processes such as expression level, are known to effect evolutionary rates. However, it has been argued that the correlated rates of evolution used to predict protein interaction explicitly includes shared evolutionary history; here we test this hypothesis. Results In order to identify the evolutionary mechanisms giving rise to the correlations between interaction proteins, we use phylogenetic methods to distinguish similarities in tree topologies from similarities in genetic distances. We use a range of datasets of interacting and non-interacting proteins from Saccharomyces cerevisiae. We find that the signal of correlated evolution between interacting proteins is predominantly a result of shared evolutionary rates, rather than similarities in tree topology, independent of evolutionary divergence. Conclusions Since interacting proteins do not have tree topologies that are more similar than the control group of non-interacting proteins, it is likely that coevolution does not contribute much to, if any, of the observed correlations. PMID:23217198

  13. Visualization of electron transfer interactions of membrane proteins

    NASA Astrophysics Data System (ADS)

    Kawato, Suguru

    1991-08-01

    To visualize electron transfer interactions of proteins in the cellular nieinbrane, we have developed a polarized laser flash-induced anisotropy decay imaging. The time-resolved anisotropy is particularly sensitive to protein-protein interactions. This technique has been successfully applied to examine formation and dissociation of electron transfer complex in adrenal cortex and liver. Electron transfer plays a significant role for steroid hormone synthesis from cholesterol in adrenalcortex and for drug metabolism in liver such as detoxification of chemical compounds. Several redox partners perticipate in dynamic electron transfer interactions. The terminal enzyme cytochrome P-450 receives electrons to activate molecular oxygen, resulting in hydroxylation of various substrates.

  14. Solution Structure of Mouse Hepatitis Virus (MHV) nsp3a and Determinants of the Interaction with MHV Nucleocapsid (N) Protein

    PubMed Central

    Keane, Sarah C.

    2013-01-01

    Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [Ka], [1.4 ± 0.3] × 106 M−1). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as Ka) of 8.1 × 103 M−1. These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N. PMID:23302895

  15. From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

    PubMed Central

    Gao, Mu; Skolnick, Jeffrey

    2009-01-01

    DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein. PMID:19343221

  16. Protein solvent and weak protein protein interactions in halophilic malate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Ebel, Christine; Faou, Pierre; Zaccai, Giuseppe

    1999-01-01

    With the aim to correlate the solvation, stability and solubility properties of halophilic malate dehydrogenase, we characterized its weak interparticle interactions by small-angle neutron scattering in various solvents. The protein concentration dependence of the apparent radius of gyration and forward scattered intensity extrapolated from Guinier plots, and thus the second virial coefficient, A2, were determined for each solvent condition. In NaCl 1M+2-methylpentane-2,4-diol 30%, a solvent that promotes protein crystallization, A2 is negative, -0.4×10 -4 ml mol g -2 and indicating attractive interactions; in ammonium sulfate 3M, in which the protein precipitates at high concentrations, A2˜0. In 2-5M NaCl, 1-3.5M NaOAc, 1-4.5M KF or 1-2M (NH 4) 2SO 4, in which the protein is very soluble, A2 is positive with a value of the order of 0.4×10 -4 ml mol g -2 which decreases with increasing salt concentration. In MgCl 2 however, A2 increases with increasing salt concentration from 0.2 to 1.3M.

  17. The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction

    PubMed Central

    Martin, Catherine; Berridge, Georgina; Higgins, Christopher F; Callaghan, Richard

    1997-01-01

    The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3±4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model. PMID:9375975

  18. Protein-Protein Interactions in high moisture-extruded meat analogs and heat-induce soy Protein Gels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two commercial soy protein isolates were made into fibrous meat analogs by high moisture extrusion or into gels by heating and cooling, at varying concentrations and/or temperatures. Protein-protein interactions by extrusion or gelation were investigated through protein solubility studies of raw an...

  19. Template-based structure modeling of protein-protein interactions

    PubMed Central

    Szilagyi, Andras; Zhang, Yang

    2014-01-01

    The structure of protein-protein complexes can be constructed by using the known structure of other protein complexes as a template. The complex structure templates are generally detected either by homology-based sequence alignments or, given the structure of monomer components, by structure-based comparisons. Critical improvements have been made in recent years by utilizing interface recognition and by recombining monomer and complex template libraries. Encouraging progress has also been witnessed in genome-wide applications of template-based modeling, with modeling accuracy comparable to high-throughput experimental data. Nevertheless, bottlenecks exist due to the incompleteness of the proteinprotein complex structure library and the lack of methods for distant homologous template identification and full-length complex structure refinement. PMID:24721449

  20. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  1. Bilayer-thickness-mediated interactions between integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  2. Global approaches to study protein-protein interactions among viruses and hosts.

    PubMed

    Mendez-Rios, Jorge; Uetz, Peter

    2010-02-01

    While high-throughput protein-protein interaction screens were first published approximately 10 years ago, systematic attempts to map interactions among viruses and hosts started only a few years ago. HIV-human interactions dominate host-pathogen interaction databases (with approximately 2000 interactions) despite the fact that probably none of these interactions have been identified in systematic interaction screens. Recently, combinations of protein interaction data with RNAi and other functional genomics data allowed researchers to model more complex interaction networks. The rapid progress in this area promises a flood of new data in the near future, with clinical applications as soon as structural and functional genomics catches up with next-generation sequencing of human variation and structure-based drug design. PMID:20143950

  3. Detecting Protein-Protein Interactions in Vesicular Stomatitis Virus Using a Cytoplasmic Yeast Two Hybrid System

    PubMed Central

    Moerdyk-Schauwecker, Megan; DeStephanis, Darla; Hastie, Eric; Grdzelishvili, Valery Z.

    2011-01-01

    Summary Protein-protein interactions play an important role in many virus-encoded functions and in virus-host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses. PMID:21320532

  4. Choosing negative examples for the prediction of protein-protein interactions

    PubMed Central

    Ben-Hur, Asa; Noble, William Stafford

    2006-01-01

    The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions. PMID:16723005

  5. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

    SciTech Connect

    Chowdhury, Saiful M.; Shi, Liang; Yoon, Hyunjin; Ansong, Charles; Rommereim, Leah M.; Norbeck, Angela D.; Auberry, Kenneth J.; Moore, R. J.; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.

    2009-02-10

    We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

  6. PPISEARCHENGINE: gene ontology-based search for protein-protein interactions.

    PubMed

    Park, Byungkyu; Cui, Guangyu; Lee, Hyunjin; Huang, De-Shuang; Han, Kyungsook

    2013-01-01

    This paper presents a new search engine called PPISearchEngine which finds protein-protein interactions (PPIs) using the gene ontology (GO) and the biological relations of proteins. For efficient retrieval of PPIs, each GO term is assigned a prime number and the relation between the terms is represented by the product of prime numbers. This representation is hidden from users but facilitates the search for the interactions of a query protein by unique prime factorisation of the number that represents the query protein. For a query protein, PPISearchEngine considers not only the GO term associated with the query protein but also the GO terms at the lower level than the GO term in the GO hierarchy, and finds all the interactions of the query protein which satisfy the search condition. In contrast, the standard keyword-matching or ID-matching search method cannot find the interactions of a protein unless the interactions involve a protein with explicit annotations. To the best of our knowledge, this search engine is the first method that can process queries like 'for protein p with GO [Formula: see text], find p's interaction partners with GO [Formula: see text]'. PPISearchEngine is freely available to academics at http://search.hpid.org/. PMID:22316075

  7. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    SciTech Connect

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-08-12

    Highlights: {yields} Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. {yields} BAI2 interaction with GIP was revealed by yeast two-hybrid assay. {yields} Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. {yields} BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, {beta}-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  8. Characterization of the interactions between protein and carbon black.

    PubMed

    Chen, Tzu-Tao; Chuang, Kai-Jen; Chiang, Ling-Ling; Chen, Chun-Chao; Yeh, Chi-Tai; Wang, Liang-Shun; Gregory, Clive; Jones, Tim; BéruBé, Kelly; Lee, Chun-Nin; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

    2014-01-15

    A considerable amount of studies have been conducted to investigate the interactions of biological fluids with nanoparticle surfaces, which exhibit a high affinity for proteins and particles. However, the mechanisms underlying these interactions have not been elucidated, particularly as they relate to human health. Using bovine serum albumin (BSA) and mice bronchoalveolar lavage fluid (BALF) as models for protein-particle conjugates, we characterized the physicochemical modifications of carbon blacks (CB) with 23nm or 65nm in diameter after protein treatment. Adsorbed BALF-containing proteins were quantified and identified by pathways, biological analyses and protein classification. Significant modifications of the physicochemistry of CB were induced by the addition of BSA. Enzyme modulators and hydrolase predominately interacted with CB, with protein-to-CB interactions that were associated with the coagulation pathways. Additionally, our results revealed that an acute-phase response could be activated by these proteins. With regard to human health, the present study revealed that the CB can react with proteins (∼55kDa and 70kDa) after inhalation and may modify the functional structures of lung proteins, leading to the activation of acute-inflammatory responses in the lungs. PMID:24291665

  9. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    PubMed Central

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  10. Visualization and targeted disruption of protein interactions in living cells

    PubMed Central

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  11. Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions

    PubMed Central

    Blazer, Levi L.; Roman, David L.; Muxlow, Molly R.; Neubig, Richard R.

    2010-01-01

    A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided. PMID:20069525

  12. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  13. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N P; Riedmayr, Lisa M; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  14. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGESBeta

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  15. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  16. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one. PMID:23112006

  17. Mutual diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    The generalized Stokes-Einstein equation is used, together with the two-dimensional pressure equation, to analyze mutual diffusion in concentrated membrane systems. These equations can be used to investigate the role that both direct and hydrodynamic interactions play in determining diffusive behavior. Here only direct interactions are explicitly incorporated into the theory at high densities; however, both direct and hydrodynamic interactions are analyzed for some dilute solutions. We look at diffusion in the presence of weak attractions, soft repulsions, and hard-core repulsions. It is found that, at low densities, attractions retard mutual diffusion while repulsions enhance it. Mechanistically, attractions tend to tether particles together and oppose the dissipation of gradients or fluctuations in concentration, while repulsions provide a driving force that pushes particles apart. At higher concentrations, changes in the structure of the fluid enhance mutual diffusion even in the presence of attractions. It is shown that the theoretical description of postelectrophoresis relaxation and fluorescence correlation spectroscopy experiments must be modified if interacting systems are studied. The effects of interactions on mutual diffusion coefficients have probably already been seen in postelectrophoresis relaxation experiments. PMID:2775829

  18. Monitoring the interaction of hydraulic fracturing fluid with Marcellus Shale using Sr isotopes: a comparison of laboratory experiments with field scale observations (Invited)

    NASA Astrophysics Data System (ADS)

    Wall, A. J.; Hakala, A.; Marcon, V.; Joseph, C.

    2013-12-01

    Strontium isotopes have the potential to be an effective tool for differentiating Marcellus Shale derived-fluids from other sources in surface and ground waters (Chapman et al. 2012, doi: 10.1021/es204005g). Water that is co-produced during gas extraction is likely influenced by fluid/rock interactions during hydraulic fracturing (HF) and monitoring changes in Sr isotope ratios can provide insight into reactions occurring within the shale formation. However, questions persist as to what controls the Sr isotopic composition of Marcellus Shale fluids, especially during HF. Here we compare laboratory experiments, simulating the dissolution of the Marcellus Shale during HF, with a time-series of water samples taken from a Marcellus Shale gas wells after HF has occurred. For the laboratory experiments, a core sample of Marcellus Shale from Greene County, PA was crushed and placed into a high P and T reaction vessel. Solutions were added in two different experiments: one with synthetic brine, and another using brine+HF fluid. The HF fluid was made up of components listed on fracfocus.org. Experiments were run for ~16 days at 27.5 MPa and 130oC. Aqueous samples were periodically removed for analysis and Sr isotope ratios were measured by MC-ICP-MS. Using just brine, the pH of the solution decreased from 7.6 to 5.3 after 24 hrs, then reached a steady state at ~6.1. Sr/Ca molar ratios in the fluid started at 2.3 after 24 hours and decreased to 1.8 over ~16 days. During this time only 6% of the total inorganic carbon (TIC) dissolved from the shale. The ɛSr values started at +43.2 and decreased to +42.4. In the experiment using brine+HF fluid, the pH started at 1.8 and rose slowly to a steady value of 5.6 by day 6. The Sr and Ca concentrations were higher than the brine experiment, but the Sr/Ca ratios remained lower at ~0.3 through the experiment. The increased Ca release, as well as the dissolution of over 60% of the TIC, suggests the dissolution of a carbonate mineral

  19. Investigation of protein-ligand interactions by mass spectrometry.

    PubMed

    Sinz, Andrea

    2007-04-01

    The rate of drug discovery is greatly dependent on the development and improvement of rapid and reliable analytical methods that allow screening for protein-ligand interactions. The solution-based methods for investigating protein-ligand interactions by mass spectrometry (MS), which are discussed in this paper, are hydrogen/deuterium exchange of protein backbone amide hydrogens, and photoaffinity labeling. Moreover, MS analysis of intact noncovalent protein-ligand complexes is described. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with its ultra-high resolution and excellent mass accuracy is also considered herein as it is gaining increasing popularity for a mass spectrometric investigation of protein-ligand interactions. PMID:17299828

  20. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

    PubMed

    Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano

    2013-02-01

    Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction. PMID:23264567

  1. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  2. Detecting Overlapping Protein Complexes by Rough-Fuzzy Clustering in Protein-Protein Interaction Networks

    PubMed Central

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  3. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  4. A protein interaction map for cell polarity development.

    PubMed

    Drees, B L; Sundin, B; Brazeau, E; Caviston, J P; Chen, G C; Guo, W; Kozminski, K G; Lau, M W; Moskow, J J; Tong, A; Schenkman, L R; McKenzie, A; Brennwald, P; Longtine, M; Bi, E; Chan, C; Novick, P; Boone, C; Pringle, J R; Davis, T N; Fields, S; Drubin, D G

    2001-08-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  5. PIPs: human protein–protein interaction prediction database

    PubMed Central

    McDowall, Mark D.; Scott, Michelle S.; Barton, Geoffrey J.

    2009-01-01

    The PIPs database (http://www.compbio.dundee.ac.uk/www-pips) is a resource for studying protein–protein interactions in human. It contains predictions of >37 000 high probability interactions of which >34 000 are not reported in the interaction databases HPRD, BIND, DIP or OPHID. The interactions in PIPs were calculated by a Bayesian method that combines information from expression, orthology, domain co-occurrence, post-translational modifications and sub-cellular location. The predictions also take account of the topology of the predicted interaction network. The web interface to PIPs ranks predictions according to their likelihood of interaction broken down by the contribution from each information source and with easy access to the evidence that supports each prediction. Where data exists in OPHID, HPRD, DIP or BIND for a protein pair this is also reported in the output tables returned by a search. A network browser is included to allow convenient browsing of the interaction network for any protein in the database. The PIPs database provides a new resource on protein–protein interactions in human that is straightforward to browse, or can be exploited completely, for interaction network modelling. PMID:18988626

  6. Biophysics of protein-DNA interactions and chromosome organization

    PubMed Central

    Marko, John F.

    2014-01-01

    The function of DNA in cells depends on its interactions with protein molecules, which recognize and act on base sequence patterns along the double helix. These notes aim to introduce basic polymer physics of DNA molecules, biophysics of protein-DNA interactions and their study in single-DNA experiments, and some aspects of large-scale chromosome structure. Mechanisms for control of chromosome topology will also be discussed. PMID:25419039

  7. Overview of the protein-protein interaction annotation extraction task of BioCreative II

    PubMed Central

    Krallinger, Martin; Leitner, Florian; Rodriguez-Penagos, Carlos; Valencia, Alfonso

    2008-01-01

    Background: The biomedical literature is the primary information source for manual protein-protein interaction annotations. Text-mining systems have been implemented to extract binary protein interactions from articles, but a comprehensive comparison between the different techniques as well as with manual curation was missing. Results: We designed a community challenge, the BioCreative II protein-protein interaction (PPI) task, based on the main steps of a manual protein interaction annotation workflow. It was structured into four distinct subtasks related to: (a) detection of protein interaction-relevant articles; (b) extraction and normalization of protein interaction pairs; (c) retrieval of the interaction detection methods used; and (d) retrieval of actual text passages that provide evidence for protein interactions. A total of 26 teams submitted runs for at least one of the proposed subtasks. In the interaction article detection subtask, the top scoring team reached an F-score of 0.78. In the interaction pair extraction and mapping to SwissProt, a precision of 0.37 (with recall of 0.33) was obtained. For associating articles with an experimental interaction detection method, an F-score of 0.65 was achieved. As for the retrieval of the PPI passages best summarizing a given protein interaction in full-text articles, 19% of the submissions returned by one of the runs corresponded to curator-selected sentences. Curators extracted only the passages that best summarized a given interaction, implying that many of the automatically extracted ones could contain interaction information but did not correspond to the most informative sentences. Conclusion: The BioCreative II PPI task is the first attempt to compare the performance of text-mining tools specific for each of the basic steps of the PPI extraction pipeline. The challenges identified range from problems in full-text format conversion of articles to difficulties in detecting interactor protein pairs and then

  8. The Intraviral Protein Interaction Network of Hepatitis C Virus*

    PubMed Central

    Hagen, Nicole; Bayer, Karen; Rösch, Kathrin; Schindler, Michael

    2014-01-01

    Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometry-based FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Schindler, M. (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One 5, e9344). HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they were functional. FACS-FRET measurements identified a total of 20 interactions; 13 of these were previously described and have now been confirmed in living cells via our method. Among the seven novel protein binding pairs, HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV. Our work demonstrates the feasibility of rapidly generating small interaction networks via FACS-FRET and defines the network of intra-HCV protein interactions. Furthermore, our data support an important role of p7 in HCV assembly. PMID:24797426

  9. Linkers in the structural biology of protein–protein interactions

    PubMed Central

    Reddy Chichili, Vishnu Priyanka; Kumar, Veerendra; Sivaraman, J

    2013-01-01

    Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly-rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly-rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein–protein complex. Gly-rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand-binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein–protein complexes, and to obtain protein dimers. PMID:23225024

  10. Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction

    PubMed Central

    van Dijk, Aalt D. J.; Morabito, Giuseppa; Fiers, Martijn; van Ham, Roeland C. H. J.; Angenent, Gerco C.; Immink, Richard G. H.

    2010-01-01

    Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution. PMID

  11. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  12. Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

    PubMed Central

    2012-01-01

    Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed. PMID:22789293

  13. Discovering Distinct Functional Modules of Specific Cancer Types Using Protein-Protein Interaction Networks

    PubMed Central

    Shen, Ru; Wang, Xiaosheng; Guda, Chittibabu

    2015-01-01

    Background. The molecular profiles exhibited in different cancer types are very different; hence, discovering distinct functional modules associated with specific cancer types is very important to understand the distinct functions associated with them. Protein-protein interaction networks carry vital information about molecular interactions in cellular systems, and identification of functional modules (subgraphs) in these networks is one of the most important applications of biological network analysis. Results. In this study, we developed a new graph theory based method to identify distinct functional modules from nine different cancer protein-protein interaction networks. The method is composed of three major steps: (i) extracting modules from protein-protein interaction networks using network clustering algorithms; (ii) identifying distinct subgraphs from the derived modules; and (iii) identifying distinct subgraph patterns from distinct subgraphs. The subgraph patterns were evaluated using experimentally determined cancer-specific protein-protein interaction data from the Ingenuity knowledgebase, to identify distinct functional modules that are specific to each cancer type. Conclusion. We identified cancer-type specific subgraph patterns that may represent the functional modules involved in the molecular pathogenesis of different cancer types. Our method can serve as an effective tool to discover cancer-type specific functional modules from large protein-protein interaction networks. PMID:26495282

  14. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  15. Quantification of protein interaction kinetics in a micro droplet

    NASA Astrophysics Data System (ADS)

    Yin, L. L.; Wang, S. P.; Shan, X. N.; Zhang, S. T.; Tao, N. J.

    2015-11-01

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  16. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling. PMID:25873628

  17. Quantification of protein interaction kinetics in a micro droplet

    SciTech Connect

    Yin, L. L.; Wang, S. P. E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J. E-mail: njtao@asu.edu; Zhang, S. T.

    2015-11-15

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  18. Ex vivo identification of protein-protein interactions involving the dopamine transporter.

    PubMed

    Hadlock, Gregory C; Nelson, Chad C; Baucum, Anthony J; Hanson, Glen R; Fleckenstein, Annette E

    2011-03-30

    The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT. PMID:21291912

  19. Gel mobility shift assays to detect protein-RNA interactions.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2012-01-01

    The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins. PMID:22736005

  20. Proteins differentially interact with grapefruit furanocoumarins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapefruit juice (GFJ) interferes with the cytochrome P450 3A4 activity responsible for metabolizing certain medications. This interference is referred to as the "grapefruit-drug interaction". Grapefruit furanocoumarins (FCs), such as 6', 7'-dihydroxybergamottin (DHB) and bergamottin (BM), have been...

  1. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations.

    PubMed

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I; Kudla, Jörg

    2014-01-01

    The analyses of protein-protein interactions are crucial for understanding cellular processes including signal transduction, protein trafficking, and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further, we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  2. Computational Prediction of Protein–Protein Interaction Networks: Algo-rithms and Resources

    PubMed Central

    Zahiri, Javad; Bozorgmehr, Joseph Hannon; Masoudi-Nejad, Ali

    2013-01-01

    Protein interactions play an important role in the discovery of protein functions and pathways in biological processes. This is especially true in case of the diseases caused by the loss of specific protein-protein interactions in the organism. The accuracy of experimental results in finding protein-protein interactions, however, is rather dubious and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. Computational methods have attracted tremendous attention among biologists because of the ability to predict protein-protein interactions and validate the obtained experimental results. In this study, we have reviewed several computational methods for protein-protein interaction prediction as well as describing major databases, which store both predicted and detected protein-protein interactions, and the tools used for analyzing protein interaction networks and improving protein-protein interaction reliability. PMID:24396273

  3. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormon...

  4. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a practical…

  5. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

    PubMed

    Scott, Emily E; Wolf, C Roland; Otyepka, Michal; Humphreys, Sara C; Reed, James R; Henderson, Colin J; McLaughlin, Lesley A; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P; Barnaba, Carlo; Brozik, James A; Jones, Jeffrey P; Estrada, D Fernando; Laurence, Jennifer S; Park, Ji Won; Backes, Wayne L

    2016-04-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  6. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  7. Identification of Redox and Glucose-Dependent Txnip Protein Interactions

    PubMed Central

    Neuharth, Skyla; Kim, Dae In; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip. PMID:27437069

  8. Measurement of Quadrupole Interactions in La1 - x Sr x CoO3 Perovskites Using TDPAC Technique

    NASA Astrophysics Data System (ADS)

    Junqueira, A. C.; Carbonari, A. W.; Saxena, R. N.; Mestnik-Filho, J.; Droga, R.

    2004-11-01

    The Perturbed Angular Correlation (PAC) technique was used to study the quadrupole interactions in the La1 - x Sr x CoO3 (0 ≤ x ≤ 0.08) perovskites using 111Cd and 181Ta probes. The radioactive parent nuclei 111In and 181Hf were introduced in the oxide lattice through chemical process during sample preparation and found to occupy only Co sites. The measurements cover a temperature range from 10 to 1150 K except the pure LaCoO3 for which an additional measurement was made at 4.2 K. The measured quadrupole frequencies were found to decrease linearly with increasing temperature as well as with increasing Sr concentration. Temperature dependence of quadrupole frequency in the pure LaCoO3 shows small discontinuities around 70 90 K and 500 600 K which have been atributed to thermally activated spin state transitions, from the low-spin (LS) ground state electronic configuration of Co+3 ion to the intermediate-spin (IS) state and from intermediate-spin (IS) state to high-spin (HS) state respectively, observed in some recent studies.

  9. Examination of Interactions of Oppositely Charged Proteins in Gels

    SciTech Connect

    Ramasamy,P.; El-Maghrabi, M.; Halada, G.; Miller, L.; Rafailovich, M.

    2007-01-01

    Understanding the interactions of proteins with one another serves as an important step for developing faster protein separation methods. To examine protein-protein interactions of oppositely charged proteins, fluorescently labeled albumin and poly-L-lysine were subjected to electrophoresis in agarose gels, in which the cationic albumin and the anionic poly-L-lysine were allowed to migrate toward each other and interact. Fluorescence microscopy was used to image fluorescently tagged proteins in the gel. The secondary structure of the proteins in solution was studied using conventional FTIR spectroscopy. Results showed that sharp interfaces were formed where FITC tagged albumin met poly-L-lysine and that the interfaces did not migrate after they had been formed. The position of the interface in the gel was found to be linearly dependent upon the relative concentration of the proteins. The formation of the interface also depended upon the fluorescent tag attached to the protein. The size of the aggregates at the interface, the fluorescence intensity modifications, and the mobility of the interface for different pore sizes of the gel were investigated. It was observed that the interface was made up of aggregates of about 1 {mu}m in size. Using dynamic light scattering, it was observed that the size of the aggregates that formed due to interactions of oppositely charged proteins depended upon the fluorescent tags attached to the proteins. The addition of small amounts of poly-L-lysine to solutions containing FITC albumin decreased the zeta potential drastically. For this, we propose a model suggesting that adding small amounts of poly-L-lysine to solutions containing FITC -albumin favors the formation of macromolecular complexes having FITC albumin molecules on its surface. Although oppositely charged FITC tagged poly-L-lysine and FITC tagged albumin influence each other's migration velocities by forming aggregates, there were no observable secondary structural

  10. ProteinShop: A tool for interactive protein manipulation and steering

    SciTech Connect

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  11. Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

    PubMed Central

    Filteau, Marie; Leducq, Jean-Baptiste; Dubé, Alexandre K.; Landry, Christian R.

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs. PMID:25867901

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

    PubMed

    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. PMID:23422566

  13. Lipid-protein interactions: Lessons learned from stress.

    PubMed

    Battle, A R; Ridone, P; Bavi, N; Nakayama, Y; Nikolaev, Y A; Martinac, B

    2015-09-01

    Biological membranes are essential for normal function and regulation of cells, forming a physical barrier between extracellular and intracellular space and cellular compartments. These physical barriers are subject to mechanical stresses. As a consequence, nature has developed proteins that are able to transpose mechanical stimuli into meaningful intracellular signals. These proteins, termed Mechanosensitive (MS) proteins provide a variety of roles in response to these stimuli. In prokaryotes these proteins form transmembrane spanning channels that function as osmotically activated nanovalves to prevent cell lysis by hypoosmotic shock. In eukaryotes, the function of MS proteins is more diverse and includes physiological processes such as touch, pain and hearing. The transmembrane portion of these channels is influenced by the physical properties such as charge, shape, thickness and stiffness of the lipid bilayer surrounding it, as well as the bilayer pressure profile. In this review we provide an overview of the progress to date on advances in our understanding of the intimate biophysical and chemical interactions between the lipid bilayer and mechanosensitive membrane channels, focusing on current progress in both eukaryotic and prokaryotic systems. These advances are of importance due to the increasing evidence of the role the MS channels play in disease, such as xerocytosis, muscular dystrophy and cardiac hypertrophy. Moreover, insights gained from lipid-protein interactions of MS channels are likely relevant not only to this class of membrane proteins, but other bilayer embedded proteins as well. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25922225

  14. Efficient detection of RNA–protein interactions using tethered RNAs

    PubMed Central

    Loiselle, David; Haystead, Timothy A.; Macara, Ian G.

    2011-01-01

    The diverse localization of transcripts in cells suggests that there are many specific RNA–protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3′-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3′-UTR. Our tRSA technique should find wide application in mapping the RNA–protein interactome. PMID:21300640

  15. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  16. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    PubMed

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  17. The Development of an Interactive Videodisc Program on Protein Synthesis.

    ERIC Educational Resources Information Center

    Hazan, Charlene Corey

    An interactive videodisk (IVD) program was developed to reinforce learning of the biological concept of protein synthesis for high school students. The laser videodisc "The Living Textbook Life Science" was the source of frames, and the authoring system of G. Smith was used to create the disc. The interactive program was designed to make the…

  18. Influence of hydrodynamic interactions on mechanical unfolding of proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, P.; Cieplak, Marek

    2007-07-01

    We incorporate hydrodynamic interactions in a structure-based model of ubiquitin and demonstrate that the hydrodynamic coupling may reduce the peak force when stretching the protein at constant speed, especially at larger speeds. Hydrodynamic interactions are also shown to facilitate unfolding at constant force and inhibit stretching by fluid flows.

  19. Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis.

    PubMed

    Huang, Chih-Ching; Cao, Zehui; Chang, Huan-Tsung; Tan, Weihong

    2004-12-01

    Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies. PMID:15571349

  20. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  1. Sequence-based prediction of protein-protein interaction sites with L1-logreg classifier.

    PubMed

    Dhole, Kaustubh; Singh, Gurdeep; Pai, Priyadarshini P; Mondal, Sukanta

    2014-05-01

    Protein-protein interactions are of central importance for virtually every process in a living cell. Information about the interaction sites in proteins improves our understanding of disease mechanisms and can provide the basis for new therapeutic approaches. Since a multitude of unique residue-residue contacts facilitate the interactions, protein-protein interaction sites prediction has become one of the most important and challenging problems of computational biology. Although much progress in this field has been reported, this problem is yet to be satisfactorily solved. Here, a novel method (LORIS: L1-regularized LOgistic Regression based protein-protein Interaction Sites predictor) is proposed, that identifies interaction residues, using sequence features and is implemented via the L1-logreg classifier. Results show that LORIS is not only quite effective, but also, performs better than existing state-of-the art methods. LORIS, available as standalone package, can be useful for facilitating drug-design and targeted mutation related studies, which require a deeper knowledge of protein interactions sites. PMID:24486250

  2. Protein-protein interaction between ezrin and p65 in human breast cancer cells.

    PubMed

    Tang, R; Li, F X; Shao, W F; Wen, Q S; Yu, X R; Xiong, J B

    2016-01-01

    Our study aimed to investigate the co-localization and protein-protein interactions between ezrin and p65 in human breast cancer cells. Liquid chromatography-mass spectrometry (LCMS) was used to uncover novel protein interactions with ezrin in MDA-MB-231 cells. Endogenous co-immunoprecipitation was used to validate protein-protein interactions between ezrin and p65 in MDA-MB-231. Exogenous interactions between ezrin and p65 were validated in MDA-MB-231 cells via Flag-ezrin and HA-p65 co-transfection and followed by co-immunoprecipitation. Immunofluorescence staining was used to visualize ezrin and p65 co-localization in MDA-MB-231. LCMS results showed that there were 1000 proteins interacting with ezrin in MDA-MB-231 cells. Ezrin and p65 interactions were confirmed with both endogenous and exogenous methods. We were also able to visualize ezrin and p65 co-localization in MDA-MB-231. In summary, we found protein-protein interactions between Ezrin and p65 in human breast cancer cells. PMID:27420986

  3. Homology Inference of Protein-Protein Interactions via Conserved Binding Sites

    PubMed Central

    Tyagi, Manoj; Thangudu, Ratna R.; Zhang, Dachuan; Bryant, Stephen H.; Madej, Thomas; Panchenko, Anna R.

    2012-01-01

    The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server) to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons). Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein. PMID:22303436

  4. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  5. Mechanisms of Peroxynitrite Interactions with Heme Proteins

    PubMed Central

    Su, Jia; Groves, John T.

    2010-01-01

    Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. In this Forum we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve nitric oxide and peroxynitrite. We have recently described direct evidence that ferrylMb and NO2 are both produced during the reaction of PN and metmyolgobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [FeIV=O .NO2]. This caged pair reacts mainly via internal return with a rate constant kr to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, kobs of heme-mediated PN decomposition. Freely-diffusing NO2, which is liberated concomitantly from the radical pair (ke), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a β-sheet structure, at the expense of turns and helices at low pH. It is proposed that a short β-sheet segment, comprising residues 37-39 and 58-61, extends itself into the large 37-61 loop when the latter is destabilized by protonation of H26, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in

  6. Experimental study of the Mg and Sr isotopic evolution of seawater interacting with basalt between 150 and 300 ° C.

    NASA Astrophysics Data System (ADS)

    Voigt, Martin; Pearce, Christopher R.; Oelkers, Eric H.

    2016-04-01

    The chemical exchange of material between seawater and the oceanic crust plays a major role in marine geochemical cycles [1]. Isotopic signatures provide an important means of tracing elemental transfer in hydrothermal environments, yet only a limited amount of experimental data on the extent of isotopic fractionation under these conditions is currently available. This study consequently investigated the extent of δ26/24Mg and 87Sr/86Sr isotopic variation during a seawater-basalt interaction experiments at 150, 250 and 290 ° C. A suite of closed system experiments were run for several months at each temperature under saturated water pressure, using either crystalline or glassy basalt as the starting material and a water/rock ratio of 10 or 25. Our results demonstrate that the dissolution of basaltic material in hydrothermal environments occurs at the same time as the precipitation of alteration minerals (mainly smectite and anhydrite), which is consistent with results from similar studies in the past (e.g. [2]). As expected, the rate of reaction using crystalline basalt was slower than with basaltic glass, and both sample types reacted faster at higher temperatures. The 87Sr/86Sr composition of the experimental fluids decreased from the initial seawater value (0.70916) towards the lower basaltic signature during the experiments (0.70317), demonstrating the progressive release of Sr during basalt dissolution. Magnesium was steadily removed from the fluid via the precipitation of clay minerals, with the residual fluids having progressively lighter δ26/24Mg compositions. The mean Mg isotope fractionation factor (αsolid‑solution) observed at 250 oC was 1.0005±0.0002, supporting low-temperature evidence that clay minerals preferentially incorporate isotopically heavy magnesium [3]. These experiments provide quantitative information on the extent of Mg isotopic fractionation between fluids and secondary silicate minerals in hydrothermal systems, and demonstrate

  7. Protein-protein interaction inference based on semantic similarity of Gene Ontology terms.

    PubMed

    Zhang, Shu-Bo; Tang, Qiang-Rong

    2016-07-21

    Identifying protein-protein interactions is important in molecular biology. Experimental methods to this issue have their limitations, and computational approaches have attracted more and more attentions from the biological community. The semantic similarity derived from the Gene Ontology (GO) annotation has been regarded as one of the most powerful indicators for protein interaction. However, conventional methods based on GO similarity fail to take advantage of the specificity of GO terms in the ontology graph. We proposed a GO-based method to predict protein-protein interaction by integrating different kinds of similarity measures derived from the intrinsic structure of GO graph. We extended five existing methods to derive the semantic similarity measures from the descending part of two GO terms in the GO graph, then adopted a feature integration strategy to combines both the ascending and the descending similarity scores derived from the three sub-ontologies to construct various kinds of features to characterize each protein pair. Support vector machines (SVM) were employed as discriminate classifiers, and five-fold cross validation experiments were conducted on both human and yeast protein-protein interaction datasets to evaluate the performance of different kinds of integrated features, the experimental results suggest the best performance of the feature that combines information from both the ascending and the descending parts of the three ontologies. Our method is appealing for effective prediction of protein-protein interaction. PMID:27117309

  8. Interaction of the tobacco lectin with histone proteins.

    PubMed

    Schouppe, Dieter; Ghesquière, Bart; Menschaert, Gerben; De Vos, Winnok H; Bourque, Stéphane; Trooskens, Geert; Proost, Paul; Gevaert, Kris; Van Damme, Els J M

    2011-03-01

    The tobacco (Nicotiana tabacum) agglutinin or Nictaba is a member of a novel class of plant lectins residing in the nucleus and the cytoplasm of tobacco cells. Since tobacco lectin expression is only observed after the plant has been subjected to stress situations such as jasmonate treatment or insect attack, Nictaba is believed to act as a signaling protein involved in the stress physiology of the plant. In this paper, a nuclear proteomics approach was followed to identify the binding partners for Nictaba in the nucleus and the cytoplasm of tobacco cv Xanthi cells. Using lectin affinity chromatography and pull-down assays, it was shown that Nictaba interacts primarily with histone proteins. Binding of Nictaba with histone H2B was confirmed in vitro using affinity chromatography of purified calf thymus histone proteins on a Nictaba column. Elution of Nictaba-interacting histone proteins was achieved with 1 m N-acetylglucosamine (GlcNAc). Moreover, mass spectrometry analyses indicated that the Nictaba-interacting histone proteins are modified by O-GlcNAc. Since the lectin-histone interaction was shown to be carbohydrate dependent, it is proposed that Nictaba might fulfill a signaling role in response to stress by interacting with O-GlcNAcylated proteins in the plant cell nucleus. PMID:21224338

  9. Interaction of ion channels and receptors with PDZ domain proteins.

    PubMed

    Kornau, H C; Seeburg, P H; Kennedy, M B

    1997-06-01

    The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo. PMID:9232802

  10. Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

    PubMed Central

    Gao, Geng; Somwar, Romel; Zhang, Zijuan; Laserson, Uri; Ciccia, Alberto; Pavlova, Natalya; Church, George; Zhang, Wei; Kesari, Santosh; Elledge, Stephen J.

    2014-01-01

    Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib. PMID:23503679

  11. Protein-lipid interactions: paparazzi hunting for snap-shots.

    PubMed

    Haberkant, Per; van Meer, Gerrit

    2009-08-01

    Photoactivatable groups meeting the criterion of minimal perturbance allow the investigation of interactions in biological samples. Here, we review the application of photoactivatable groups in lipids enabling the study of protein-lipid interactions in (biological) membranes. The chemistry of various photoactivatable groups is summarized and the specificity of the interactions detected is discussed. The recent introduction of 'click chemistry' in photocrosslinking of membrane proteins by photo-activatable lipids opens new possibilities for the analysis of crosslinked products and will help to close the gap between proteomics and lipidomics. PMID:19426134

  12. Hexagonal pnictide SrPtAs; the role of spin-orbit interaction and locally broken inversion symmetry

    NASA Astrophysics Data System (ADS)

    Rhim, S. H.; Youn, S. J.; Fischer, M. H.; Agterberg, D. F.; Sigrist, M.; Weinert, M.; Freeman, A. J.

    2012-02-01

    The first hexagonal pnictide superconductor SrPtAs which consists of stacked PtAs layers has been studied using the FLAPW methodootnotetextWimmer, Krakauer, Weinert, and Freeman, Phys.Rev.B. 24, 864 (1981) and tight-binding methods. The single PtAs layer forms a honeycomb structure that exhibits: (1) locally broken inversion symmetry despite the presence of the global inversion center, and (2) strong spin-orbit interaction, for which physical consequences are nontrivial. Based on these findings, we predict significant enhancement of both the spin susceptibility and the paramagnetic limiting field with respect to the usual s wave superconductors. Further, we suggest an increase of TC by electron doping of a van Hove singularity.

  13. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  14. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  15. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-04-01

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications. PMID:26942693

  16. Structural studies of lipid-protein interactions on cushioned bilayers

    NASA Astrophysics Data System (ADS)

    Ghosh, S. K.; Mukhopadhyay, M. K.; Ma, Y.; Lopez, I.; Bera, S.; Lurio, L. B.; Chakrabarti, A.; Kim, J. E.; Sanyal, M. K.; Sinha, S. K.

    2013-03-01

    Biological membranes are heterogeneous and dynamical organizations of lipids and proteins, which perform functions fundamental to cell survival. Lipid-protein interactions control these functions by influencing folding and stability of integral or peripheral membrane proteins. Further, the incorporation or adsorption of these proteins into the membrane can in turn influence the lipid bilayer properties. In spite of some progress in understanding this process, a detailed structural analysis is lacking. Towards a better understanding of this interaction, we have performed an advanced interface sensitive scattering experiment using synchrotron x-rays. To accurately mimic the biological membranes with their natural thermal fluctuations and in-plane mobility of lipid molecules, polymer cushioned lipid bilayers have been used. This study shows that the adsorption of peripheral membrane proteinspectrindepends on the lipid headgroups, exhibiting different types of binding to phosphatidylcholine (PC) and phosphatidylethanolamie (PE). Further, the interaction of outer membrane protein A (OMP-A), an integral membrane protein is sensitive to the thermodynamic phase of the lipids. A detailed physical modeling of the lipid-protein interactions is under way.

  17. On the surface interactions of proteins with lignin.

    PubMed

    Salas, Carlos; Rojas, Orlando J; Lucia, Lucian A; Hubbe, Martin A; Genzer, Jan

    2013-01-01

    Lignins are used often in formulations involving proteins but little is known about the surface interactions between these important biomacromolecules. In this work, we investigate the interactions at the solid-liquid interface of lignin with the two main proteins in soy, glycinin (11S) and β-conglycinin (7S). The extent of adsorption of 11S and 7S onto lignin films and the degree of hydration of the interfacial layers is quantified via Quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR). Solution ionic strength and protein denaturation (2-mercaptoethanol and urea) critically affect the adsorption process as protein molecules undergo conformational changes and their hydrophobic or hydrophilic amino acid residues interact with the surrounding medium. In general, the adsorption of the undenatured proteins onto lignin is more extensive compared to that of the denatured biomolecules and a large amount of water is coupled to the adsorbed molecules. The reduction in water contact angle after protein adsorption (by ~40° and 35° for undenatured 11S and 7S, respectively) is explained by strong nonspecific interactions between soy proteins and lignin. PMID:23234476

  18. A selection that reports on protein-protein interactions within a thermophilic bacterium.

    PubMed

    Nguyen, Peter Q; Silberg, Jonathan J

    2010-07-01

    Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions. PMID:20418388

  19. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  20. Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors.

    PubMed

    Bornert, Olivier; Møller, Thor C; Boeuf, Julien; Candusso, Marie-Pierre; Wagner, Renaud; Martinez, Karen L; Simonin, Frederic

    2013-01-01

    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs. PMID:23441177

  1. Interactions between RNase P protein subunits in Archaea

    PubMed Central

    Hall, Thomas A.; Brown, James W.

    2004-01-01

    A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeon Methanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed by HIS3 and LacZ reporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme. PMID:15810434

  2. MEGADOCK: an all-to-all protein-protein interaction prediction system using tertiary structure data.

    PubMed

    Ohue, Masahito; Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ishida, Takashi; Akiyama, Yutaka

    2014-01-01

    The elucidation of protein-protein interaction (PPI) networks is important for understanding cellular structure and function and structure-based drug design. However, the development of an effective method to conduct exhaustive PPI screening represents a computational challenge. We have been investigating a protein docking approach based on shape complementarity and physicochemical properties. We describe here the development of the protein-protein docking software package "MEGADOCK" that samples an extremely large number of protein dockings at high speed. MEGADOCK reduces the calculation time required for docking by using several techniques such as a novel scoring function called the real Pairwise Shape Complementarity (rPSC) score. We showed that MEGADOCK is capable of exhaustive PPI screening by completing docking calculations 7.5 times faster than the conventional docking software, ZDOCK, while maintaining an acceptable level of accuracy. When MEGADOCK was applied to a subset of a general benchmark dataset to predict 120 relevant interacting pairs from 120 x 120 = 14,400 combinations of proteins, an F-measure value of 0.231 was obtained. Further, we showed that MEGADOCK can be applied to a large-scale protein-protein interaction-screening problem with accuracy better than random. When our approach is combined with parallel high-performance computing systems, it is now feasible to search and analyze protein-protein interactions while taking into account three-dimensional structures at the interactome scale. MEGADOCK is freely available at http://www.bi.cs.titech.ac.jp/megadock. PMID:23855673

  3. 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.

  4. Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent). PMID:26157620

  5. Predicting direct protein interactions from affinity purification mass spectrometry data

    PubMed Central

    2010-01-01

    Background Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest. Results We first propose a simple probabilistic model for the interactions captured by AP-MS experiments, under which the problem of separating direct interactions from indirect ones is formulated. Then, given idealized quantitative AP-MS data, we study the problem of identifying the most likely set of direct interactions that produced the observed data. We address this challenging graph theoretical problem by first characterizing signatures that can identify weakly connected nodes as well as dense regions of the network. The rest of the direct PPI network is then inferred using a genetic algorithm. Our algorithm shows good performance on both simulated and biological networks with very high sensitivity and specificity. Then the algorithm is used to predict direct interactions from a set of AP-MS PPI data from yeast, and its performance is measured against a high-quality interaction dataset. Conclusions As the sensitivity of AP-MS pipeline improves, the fraction of indirect interactions detected will also increase, thereby making the ability to distinguish them even more desirable. Despite the simplicity of our model for indirect interactions, our method provides a good performance on the test networks. PMID:21034440

  6. Analysis of interactions between ribosomal proteins and RNA structural motifs

    PubMed Central

    2010-01-01

    Background One important goal of structural bioinformatics is to recognize and predict the interactions between protein binding sites and RNA. Recently, a comprehensive analysis of ribosomal proteins and their interactions with rRNA has been done. Interesting results emerged from the comparison of r-proteins within the small subunit in T. thermophilus and E. coli, supporting the idea of a core made by both RNA and proteins, conserved by evolution. Recent work showed also that ribosomal RNA is modularly composed. Motifs are generally single-stranded sequences of consecutive nucleotides (ssRNA) with characteristic folding. The role of these motifs in protein-RNA interactions has been so far only sparsely investigated. Results This work explores the role of RNA structural motifs in the interaction of proteins with ribosomal RNA (rRNA). We analyze composition, local geometries and conformation of interface regions involving motifs such as tetraloops, kink turns and single extruded nucleotides. We construct an interaction map of protein binding sites that allows us to identify the common types of shared 3-D physicochemical binding patterns for tetraloops. Furthermore, we investigate the protein binding pockets that accommodate single extruded nucleotides either involved in kink-turns or in arbitrary RNA strands. This analysis reveals a new structural motif, called tripod. It corresponds to small pockets consisting of three aminoacids arranged at the vertices of an almost equilateral triangle. We developed a search procedure for the recognition of tripods, based on an empirical tripod fingerprint. Conclusion A comparative analysis with the overall RNA surface and interfaces shows that contact surfaces involving RNA motifs have distinctive features that may be useful for the recognition and prediction of interactions. PMID:20122215

  7. A protein interaction map for cell polarity development

    PubMed Central

    Drees, Becky L.; Sundin, Bryan; Brazeau, Elizabeth; Caviston, Juliane P.; Chen, Guang-Chao; Guo, Wei; Kozminski, Keith G.; Lau, Michelle W.; Moskow, John J.; Tong, Amy; Schenkman, Laura R.; McKenzie, Amos; Brennwald, Patrick; Longtine, Mark; Bi, Erfei; Chan, Clarence; Novick, Peter; Boone, Charles; Pringle, John R.; Davis, Trisha N.; Fields, Stanley; Drubin, David G.

    2001-01-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  8. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  9. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host. PMID:27464694

  10. Analytical Strategies for Detecting Nanoparticle-Protein Interactions

    PubMed Central

    Li, Liwen; Mu, Qingxin; Zhang, Bin; Yan, Bing

    2016-01-01

    A significant increase in biomedical applications of nanomaterials and their potential toxicity demand versatile analytical techniques to determine protein – nanoparticle (NP) interactions. These diverse analytical techniques are reviewed. Spectroscopic methods play a significant role in studying binding affinity, binding ratio, and binding mechanisms. To elucidate NP-proteome interactions, chromatography and electrophoresis techniques are applied to separate NP-bound proteins and Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify these proteins. Since NP-protein binding is a dynamic event, Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM) are methods of choice to study kinetics of NP-protein binding. PMID:20502814

  11. Structural approaches to probing metal interaction with proteins.

    PubMed

    Parker, Lorien J; Ascher, David B; Gao, Chen; Miles, Luke A; Harris, Hugh H; Parker, Michael W

    2012-10-01

    In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein. PMID:22437159

  12. SPARC (secreted protein acidic and rich in cysteine) of the intestinal nematode Strongyloides ratti is involved in mucosa-associated parasite-host interaction.

    PubMed

    Anandarajah, Emmanuela M; Ditgen, Dana; Hansmann, Jan; Erttmann, Klaus D; Liebau, Eva; Brattig, Norbert W

    2016-06-01

    The secreted protein acidic and rich in cysteine (SPARC), found in the excretory/secretory products of Strongyloides ratti, is most strongly expressed in parasitic females. Since SPARC proteins are involved in the modulation of cell-matrix interactions, a role of the secreted S. ratti SPARC (Sr-SPARC) in the manifestation of the parasite in the host's intestine is postulated. The full-length cDNA of Sr-SPARC was identified and the protein was recombinantly expressed. The purified protein was biologically active, able to bind calcium, and to attach to mucosa-associated human cells. Addition of Sr-SPARC to an in vitro mucosal three-dimensional-cell culture model led to a time-dependent release of the cytokines TNF-α, IL-22, IL-10 and TSLP. Of importance, exposure with Sr-SPARC fostered wound closure in an intestinal epithelial cell model. Here, we demonstrate for the first time that SPARC released from the nematode is a multifunctional protein affecting the mucosal immune system. PMID:27268729

  13. Simulation Studies of Protein and Small Molecule Interactions and Reaction.

    PubMed

    Yang, L; Zhang, J; Che, X; Gao, Y Q

    2016-01-01

    Computational studies of protein and small molecule (protein-ligand/enzyme-substrate) interactions become more and more important in biological science and drug discovery. Computer modeling can provide molecular details of the processes such as conformational change, binding, and transportation of small molecules/proteins, which are not easily to be captured in experiments. In this chapter, we discussed simulation studies of both protein and small molecules from three aspects: conformation sampling, transportations of small molecules in enzymes, and enzymatic reactions involving small molecules. Both methodology developments and examples of simulation studies in this field were presented. PMID:27497167

  14. Predicting protein functions from redundancies in large-scale protein interaction networks

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj Pratim; Liang, Shoudan

    2003-01-01

    Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

  15. Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-02-01

    Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment. PMID:26689634

  16. Split-Cre recombinase effectively monitors protein-protein interactions in living bacteria.

    PubMed

    O'Brien, Sean P; DeLisa, Matthew P

    2014-03-01

    The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split-Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein-protein interactions has not been reported. In this work, we developed a protein-fragment complementation assay (PCA) based on split-Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual-fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split-Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high-affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split-Cre PCA will become a highly complementary and useful new addition to the protein-protein interaction toolbox. PMID:24390935

  17. Protein-protein interactions prediction based on iterative clique extension with gene ontology filtering.

    PubMed

    Yang, Lei; Tang, Xianglong

    2014-01-01

    Cliques (maximal complete subnets) in protein-protein interaction (PPI) network are an important resource used to analyze protein complexes and functional modules. Clique-based methods of predicting PPI complement the data defection from biological experiments. However, clique-based predicting methods only depend on the topology of network. The false-positive and false-negative interactions in a network usually interfere with prediction. Therefore, we propose a method combining clique-based method of prediction and gene ontology (GO) annotations to overcome the shortcoming and improve the accuracy of predictions. According to different GO correcting rules, we generate two predicted interaction sets which guarantee the quality and quantity of predicted protein interactions. The proposed method is applied to the PPI network from the Database of Interacting Proteins (DIP) and most of the predicted interactions are verified by another biological database, BioGRID. The predicted protein interactions are appended to the original protein network, which leads to clique extension and shows the significance of biological meaning. PMID:24578640

  18. West Nile virus methyltransferase domain interacts with protein kinase G

    PubMed Central

    2013-01-01

    Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication. PMID:23876037

  19. Inhibition of Protein-Protein Interactions and Signaling by Small Molecules

    NASA Astrophysics Data System (ADS)

    Freire, Ernesto

    2010-03-01

    Protein-protein interactions are at the core of cell signaling pathways as well as many bacterial and viral infection processes. As such, they define critical targets for drug development against diseases such as cancer, arthritis, obesity, AIDS and many others. Until now, the clinical inhibition of protein-protein interactions and signaling has been accomplished with the use of antibodies or soluble versions of receptor molecules. Small molecule replacements of these therapeutic agents have been extremely difficult to develop; either the necessary potency has been hard to achieve or the expected biological effect has not been obtained. In this presentation, we show that a rigorous thermodynamic approach that combines differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) provides a unique platform for the identification and optimization of small molecular weight inhibitors of protein-protein interactions. Recent advances in the development of cell entry inhibitors of HIV-1 using this approach will be discussed.

  20. Tuning protein-protein interactions using cosolvents: specific effects of ionic and non-ionic additives on protein phase behavior.

    PubMed

    Hansen, Jan; Platten, Florian; Wagner, Dana; Egelhaaf, Stefan U

    2016-04-21

    Cosolvents are routinely used to modulate the (thermal) stability of proteins and, hence, their interactions with proteins have been studied intensely. However, less is known about their specific effects on protein-protein interactions, which we characterize in terms of the protein phase behavior. We analyze the phase behavior of lysozyme solutions in the presence of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), glycerol, and dimethyl sulfoxide (DMSO). We experimentally determined the crystallization boundary (XB) and, in combination with data on the cloud-point temperatures (CPTs), the crystallization gap. In agreement with other studies, our data indicate that the additives might affect the protein phase behavior through electrostatic screening and additive-specific contributions. At high salt concentrations, where electrostatic interactions are screened, both the CPT and the XB are found to be linear functions of the additive concentration. Their slopes quantify the additive-specific changes of the phase behavior and thus of the protein-protein interactions. While the specific effect of NaCl is to induce attractions between proteins, DMSO, glycerol and GuHCl (with increasing strength) weaken attractions and/or induce repulsions. Except for DMSO, changes of the CPT are stronger than those of the XB. Furthermore, the crystallization gap widens in the case of GuHCl and glycerol and narrows in the case of NaCl. We relate these changes to colloidal interaction models, namely square-well and patchy interactions. PMID:27020538

  1. Suppression of collisional shifts via strong inter-atomic interactions in a 87Sr optical lattice clock

    NASA Astrophysics Data System (ADS)

    Martin, Michael; Swallows, Matthew; Bishof, Michael; Lin, Yige; Blatt, Sebastian; Rey, Ana Maria; Ye, Jun

    2011-05-01

    Optical lattice clocks based on ensembles of neutral atoms have the potential to operate at the highest levels of stability due to the parallel interrogation of many atoms. However, the control of systematic shifts in these systems is correspondingly difficult due to the potential of collisional shifts. Clocks based on ultracold fermionic ensembles still exhibit these density-dependent shifts due to a loss of indistinguishability during the clock excitation process, limiting clock accuracy. By tightly confining samples of ultracold fermionic 87Sr atoms in a two-dimensional optical lattice, as opposed to the conventional one-dimensional geometry, we increase the collisional interaction energy to be the largest relevant energy scale, thus entering the strongly interacting regime of clock operation. We show both theoretically and experimentally that this increase in interaction energy results in a paradoxical decrease in the collisional shift, reducing this key systematic to the 10-17 level. This work was supported by the ARO with funding from the DARPA OLE program, NIST, NSF, and AFOSR.

  2. Fluorous photoaffinity labeling to probe protein-small molecule interactions.

    PubMed

    Huang, Weigang; Zhang, Qisheng

    2015-01-01

    Identifying cellular targets of bioactive small molecules is essential for their applications as chemical probes or drug candidates. Of equal importance is to determine their "off-target" interactions, which usually account for unwanted properties including toxicity. Among strategies to profile small molecule-interacting proteins, photoaffinity labeling has been widely used because of its distinct advantages such as sensitivity. When combined with mass spectrometry, this approach can provide additional structural and mechanistic information, such as drug-target stoichiometry and exact interacting amino acid residues. We have described a novel fluorous photoaffinity labeling approach, in which a fluorous tag is incorporated into the photoaffinity labeling reagent to enable the enrichment of the labeled species from complex mixtures for analysis. This new feature likely makes the fluorous photoaffinity labeling approach suitable to identify transient interactions, and low-abundant, low-affinity interacting proteins in a cellular environment. PMID:25618351

  3. Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

    PubMed Central

    Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

    2006-01-01

    Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

  4. Role of Anisotropic Interactions for Proteins and Patchy Nanoparticles

    PubMed Central

    2015-01-01

    Protein–protein interactions are inherently anisotropic to some degree, with orientation-dependent interactions between repulsive and attractive or complementary regions or “patches” on adjacent proteins. In some cases it has been suggested that such patch–patch interactions dominate the thermodynamics of dilute protein solutions, as captured by the osmotic second virial coefficient (B22), but delineating when this will or will not be the case remains an open question. A series of simplified but exactly solvable models are first used to illustrate that a delicate balance exists between the strength of attractive patch–patch interactions and the patch size, and that repulsive patch–patch interactions contribute significantly to B22 for only those conditions where the repulsions are long-ranged. Finally, B22 is reformulated, without approximations, in terms of the density of states for a given interaction energy and particle–particle distance. Doing so illustrates the inherent balance of entropic and energetic contributions to B22. It highlights that simply having strong patch–patch interactions will only cause anisotropic interactions to dominate B22 solution properties if the unavoidable entropic penalties are overcome, which cannot occur if patches are too small. The results also indicate that the temperature dependence of B22 may be a simple experimental means to assess whether a small number of strongly attractive configurations dominate the dilute solution behavior. PMID:25302767

  5. Improved silicon nanowire field-effect transistors for fast protein-protein interaction screening.

    PubMed

    Lin, Ti-Yu; Li, Bor-Ran; Tsai, Sheng-Ta; Chen, Chien-Wei; Chen, Chung-Hsuan; Chen, Yit-Tsong; Pan, Chien-Yuan

    2013-02-21

    Understanding how proteins interact with each other is the basis for studying the biological mechanisms behind various physiological activities. Silicon nanowire field-effect transistors (SiNW-FETs) are sensitive sensors used to detect biomolecular interactions in real-time. However, the majority of the applications that use SiNW-FETs are for known interactions between different molecules. To explore the capability of SiNW-FETs as fast screening devices to identify unknown interacting molecules, we applied mass spectrometry (MS) to analyze molecules reversibly bound to the SiNW-FETs. Calmodulin (CaM) is a Ca(2+)-sensing protein that is ubiquitously expressed in cells and its interaction with target molecules is Ca(2+)-dependent. By modifying the SiNW-FET surface with glutathione, glutathione S-transferase (GST)-tagged CaM binds reversibly to the SiNW-FET. We first verified the Ca(2+)-dependent interaction between GST-CaM and purified troponin I, which is involved in muscle contraction, through the conductance changes of the SiNW-FET. Furthermore, the cell lysate containing overexpressed Ca(2+)/CaM-dependent protein kinase IIα induced a conductance change in the GST-CaM-modified SiNW-FET. The bound proteins were eluted and subsequently identified by MS as CaM and kinase. In another example, candidate proteins from neuronal cell lysates interacting with calneuron I (CalnI), a CaM-like protein, were captured with a GST-CalnI-modified SiNW-FET. The proteins that interacted with CalnI were eluted and verified by MS. The Ca(2+)-dependent interaction between GST-CalnI and one of the candidates, heat shock protein 70, was re-confirmed via the SiNW-FET measurement. Our results demonstrate the effectiveness of combining MS with SiNW-FETs to quickly screen interacting molecules from cell lysates. PMID:23235921

  6. Cluster conservation as a novel tool for studying protein-protein interactions evolution.

    PubMed

    Rahat, Ofer; Yitzhaky, Assif; Schreiber, Gideon

    2008-05-01

    Protein-protein interactions networks has come to be a buzzword associated with nets containing edges that represent a pair of interacting proteins (e.g. hormone-receptor, enzyme-inhibitor, antigen-antibody, and a subset of multichain biological machines). Yet, each such interaction composes its own unique network, in which vertices represent amino acid residues, and edges represent atomic contacts. Recent studies have shown that analyses of the data encapsulated in these detailed networks may impact predictions of structure-function correlation. Here, we study homologous families of protein-protein interfaces, which share the same fold but vary in sequence. In this context, we address what properties of the network are shared among relatives with different sequences (and hence different atomic interactions) and which are not. Herein, we develop the general mathematical framework needed to compare the modularity of homologous networks. We then apply this analysis to the structural data of a few interface families, including hemoglobin alpha-beta, growth hormone-receptor, and Serine protease-inhibitor. Our results suggest that interface modularity is an evolutionarily conserved property. Hence, protein-protein interfaces can be clustered down to a few modules, with the boundaries being evolutionarily conserved along homologous complexes. This suggests that protein engineering of protein-protein binding sites may be simplified by varying each module, but retaining the overall modularity of the interface. PMID:17972288

  7. Electrochemical probe for the monitoring of DNA-protein interactions.

    PubMed

    Meunier-Prest, Rita; Bouyon, Alice; Rampazzi, Eve; Raveau, Suzanne; Andreoletti, Pierre; Cherkaoui-Malki, Mustapha

    2010-08-15

    Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower than the calculated K(D) for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K(D) from 0.46 microM to more than 25 microM. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range. PMID:20447818

  8. Phospho-tyrosine dependent protein–protein interaction network

    PubMed Central

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  9. Physical Interaction between Replication Protein A (RPA) and MRN

    PubMed Central

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic decreases the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates RPA interactions with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 show that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage. PMID:19586055

  10. Constraints imposed by nonfunctional protein-protein interactions on gene expression and proteome size

    NASA Astrophysics Data System (ADS)

    Zhang, Jingshan; Maslov, Sergei; Shakhnovich, Eugene

    2009-03-01

    Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing nonfunctional interactions with promiscuous nonfunctional partners. Here we show how nonfunctional interactions limit the proteome diversity and the average concentration of co-expressed and co-localized proteins. We use yeast compartments to verify our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, while keeping individual protein concentrations sufficiently low to reduce nonfunctional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity, and differentiation.

  11. A basement-interacted fluid in the N81 deposit, Pine Point Pb-Zn District, Canada: Sr isotopic analyses of single dolomite crystals

    NASA Astrophysics Data System (ADS)

    Gromek, Paulina; Gleeson, Sarah A.; Simonetti, Antonio

    2012-10-01

    The Mississippi Valley-type Pb-Zn deposits of the Pine Point district (Northwest Territories, Canada) are located close to the eastern edge of the present day Western Canadian Sedimentary Basin. The deposits are thought to have formed as the result of basin-wide fluid flow in the Presqu'ile Barrier, the host to the ore deposits. A laser ablation multi-collector inductively coupled plasma mass spectrometric study of 87Sr/86Sr ratios of ore-related dolomites from the N81 deposit at Pine Point indicates that at least two sources of Sr were present in the mineralizing system. One fluid has a range in Sr isotopic values from 0.07073 to 0.71200 and is interpreted to be derived from Middle Devonian seawater that interacted with clastic units in the basin. The second fluid has higher Sr isotopic values (up to 0.71520), similar to those found in some Canadian Shield brines, and is interpreted to represent an evaporated seawater-derived brine which has interacted with crystalline basement rocks. Reactivation of old structures in the basement may have provided a pathway for cross-formation fluid flow to the site of mineralization. The data suggest that the stratigraphic location of the Pine Point District, near the interface between the Western Canadian Sedimentary Basin and its basement, may have exerted a fundamental control on the formation of these deposits.

  12. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  13. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  14. Electrostatic Interactions in the Denatured State Ensemble: Their Effect Upon Protein Folding and Protein Stability

    PubMed Central

    Sato, Satoshi; Horng, Jia-Cherng; Anil, Burcu

    2009-01-01

    It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol−1 to the stability of the DSE. PMID:17900519

  15. Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody.

    PubMed

    Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

    2015-01-01

    Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured

  16. Modeling of metal interaction geometries for protein-ligand docking.

    PubMed

    Seebeck, Birte; Reulecke, Ingo; Kämper, Andreas; Rarey, Matthias

    2008-05-15

    The accurate modeling of metal coordination geometries plays an important role for structure-based drug design applied to metalloenzymes. For the development of a new metal interaction model, we perform a statistical analysis of metal interaction geometries that are relevant to protein-ligand complexes. A total of 43,061 metal sites of the Protein Data Bank (PDB), containing amongst others magnesium, calcium, zinc, iron, manganese, copper, cadmium, cobalt, and nickel, were evaluated according to their metal coordination geometry. Based on statistical analysis, we derived a model for the automatic calculation and definition of metal interaction geometries for the purpose of molecular docking analyses. It includes the identification of the metal-coordinating ligands, the calculation of the coordination geometry and the superposition of ideal polyhedra to identify the optimal positions for free coordination sites. The new interaction model was integrated in the docking software FlexX and evaluated on a data set of 103 metalloprotein-ligand complexes, which were extracted from the PDB. In a first step, the quality of the automatic calculation of the metal coordination geometry was analyzed. In 74% of the cases, the correct prediction of the coordination geometry could be determined on the basis of the protein structure alone. Secondly, the new metal interaction model was tested in terms of predicting protein-ligand complexes. In the majority of test cases, the new interaction model resulted in an improved docking accuracy of the top ranking placements. PMID:18041759

  17. Redesign of a Protein-peptide Interaction: Characterization and Applications

    SciTech Connect

    Jackrel, M.; Valverde, R; Regan, L

    2009-01-01

    The design of protein-peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein-peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T-Mods (TPR-based recognition module), for antibodies in both detection and purification applications. The new protein-peptide pair has a dissociation constant that is weaker than typical antibody-antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T-Mod-peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.

  18. Machine Learning of Protein Interactions in Fungal Secretory Pathways.

    PubMed

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja; Rousu, Juho

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  19. Machine Learning of Protein Interactions in Fungal Secretory Pathways

    PubMed Central

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker’s yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  20. Interactions between Phage-Shock Proteins in Escherichia coli

    PubMed Central

    Adams, Hendrik; Teertstra, Wieke; Demmers, Jeroen; Boesten, Rolf; Tommassen, Jan

    2003-01-01

    Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor σ54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions. PMID:12562786

  1. A residue level protein-protein interaction model in electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Song, Xueyu

    2014-03-01

    The osmotic second virial coefficients B2 are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B2 of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended Fast Multipole Methods in combination with the boundary element formulation. Overall, the calculated B2 are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations of protein complexes it is demonstrated that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.

  2. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  3. Dynamics and mechanism of ultrafast water–protein interactions

    PubMed Central

    Qin, Yangzhong; Wang, Lijuan; Zhong, Dongping

    2016-01-01

    Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship. PMID:27339138

  4. Dynamics and mechanism of ultrafast water-protein interactions.

    PubMed

    Qin, Yangzhong; Wang, Lijuan; Zhong, Dongping

    2016-07-26

    Protein hydration is essential to its structure, dynamics, and function, but water-protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship. PMID:27339138

  5. Effects of Be, Sr, Fe and Mg interactions on the microstructure and mechanical properties of aluminum based aeronautical alloys

    NASA Astrophysics Data System (ADS)

    Ibrahim, Mohamed Fawzy

    dissolution, thinning, necking and fragmentation of these platelets upon increasing the solutionizing time. The pi-phase was observed to dissolve and/or transform into a cluster of very fine beta-phase platelets. In the as-cast conditions, increasing the Mg content leads to increased transformation of beta-phase platelets into Chinese-script pi-phase, regardless of the Fe content. This, in turn, decreases the harmful effect of the beta-phase. Increasing the solutionizing time leads to a decomposition of the pi-phase to the beta-phase, fragmentation of the beta-phase and spheroidization of both the eutectic Si and the pi-phase particles, thus improving alloy tensile properties. Two mechanisms of Mg2Si precipitate coarsening were observed to occur: (1) Ostwald ripening in the solution heat-treated samples and (2) clustering. Coarsening increases with increased solution heat treatment time, increased aging time, as well as with greater Mg contents. Increased Fe levels decrease the alloy quality index (Q) values, whereas adding Mg increases them. Introducing Be, in spite of it being a toxic material, Sr, or both, simultaneously improves the alloy quality index values, regardless of solutionizing time or Fe and Mg levels. Quality index values increase with solution heat treatment time from 5 to 12 hours. Higher Mg contents lead to an increase in alloy ductility, ultimate tensile strength (UTS) and yield strength (YS), while higher Fe levels can drastically decrease these properties. For the same levels of Fe and/or Mg, Be and Sr have significant effects in improving alloy mechanical properties; these effects can be readily observed in low levels of Fe and high Mg contents. Beryllium addition is beneficial in the case of high Fe contents as it lowers the harmful effects of Fe-phases in Al-Si alloys. In the case of high Fe contents, it seems that the addition of 500 ppm of Be is not sufficient for all interactions with other alloying elements. During the melting process the

  6. Computational prediction of virus-human protein-protein interactions using embedding kernelized heterogeneous data.

    PubMed

    Nourani, Esmaeil; Khunjush, Farshad; Durmuş, Saliha

    2016-05-24

    Pathogenic microorganisms exploit host cellular mechanisms and evade host defense mechanisms through molecular pathogen-host interactions (PHIs). Therefore, comprehensive analysis of these PHI networks should be an initial step for developing effective therapeutics against infectious diseases. Computational prediction of PHI data is gaining increasing demand because of scarcity of experimental data. Prediction of protein-protein interactions (PPIs) within PHI systems can be formulated as a classification problem, which requires the knowledge of non-interacting protein pairs. This is a restricting requirement since we lack datasets that report non-interacting protein pairs. In this study, we formulated the "computational prediction of PHI data" problem using kernel embedding of heterogeneous data. This eliminates the abovementioned requirement and enables us to predict new interactions without randomly labeling protein pairs as non-interacting. Domain-domain associations are used to filter the predicted results leading to 175 novel PHIs between 170 human proteins and 105 viral proteins. To compare our results with the state-of-the-art studies that use a binary classification formulation, we modified our settings to consider the same formulation. Detailed evaluations are conducted and our results provide more than 10 percent improvements for accuracy and AUC (area under the receiving operating curve) results in comparison with state-of-the-art methods. PMID:27072625

  7. Evolution and protein interactions of AP2 proteins in Brassicaceae: Evidence linking development and environmental responses.

    PubMed

    Zeng, Liping; Yin, Yue; You, Chenjiang; Pan, Qianli; Xu, Duo; Jin, Taijie; Zhang, Bailong; Ma, Hong

    2016-06-01

    Plants have evolved a large number of transcription factors (TF), which are enriched among duplicate genes, highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes, we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition, the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A. thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins, with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an anther-defective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses, including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study. PMID:26472270

  8. Partner-Aware Prediction of Interacting Residues in Protein-Protein Complexes from Sequence Data

    PubMed Central

    Ahmad, Shandar; Mizuguchi, Kenji

    2011-01-01

    Computational prediction of residues that participate in protein-protein interactions is a difficult task, and state of the art methods have shown only limited success in this arena. One possible problem with these methods is that they try to predict interacting residues without incorporating information about the partner protein, although it is unclear how much partner information could enhance prediction performance. To address this issue, the two following comparisons are of crucial significance: (a) comparison between the predictability of inter-protein residue pairs, i.e., predicting exactly which residue pairs interact with each other given two protein sequences; this can be achieved by either combining conventional single-protein predictions or making predictions using a new model trained directly on the residue pairs, and the performance of these two approaches may be compared: (b) comparison between the predictability of the interacting residues in a single protein (irrespective of the partner residue or protein) from conventional methods and predictions converted from the pair-wise trained model. Using these two streams of training and validation procedures and employing similar two-stage neural networks, we showed that the models trained on pair-wise contacts outperformed the partner-unaware models in predicting both interacting pairs and interacting single-protein residues. Prediction performance decreased with the size of the conformational change upon complex formation; this trend is similar to docking, even though no structural information was used in our prediction. An example application that predicts two partner-specific interfaces of a protein was shown to be effective, highlighting the potential of the proposed approach. Finally, a preliminary attempt was made to score docking decoy poses using prediction of interacting residue pairs; this analysis produced an encouraging result. PMID:22194998

  9. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  10. An analysis pipeline for the inference of protein-protein interaction networks

    SciTech Connect

    Taylor, Ronald C.; Singhal, Mudita; Daly, Don S.; Gilmore, Jason M.; Cannon, William R.; Domico, Kelly O.; White, Amanda M.; Auberry, Deanna L.; Auberry, Kenneth J.; Hooker, Brian S.; Hurst, G. B.; McDermott, Jason E.; McDonald, W. H.; Pelletier, Dale A.; Schmoyer, Denise A.; Wiley, H. S.

    2009-12-01

    An analysis pipeline has been created for deployment of a novel algorithm, the Bayesian Estimator of Protein-Protein Association Probabilities (BEPro), for use in the reconstruction of protein-protein interaction networks. We have combined the Software Environment for BIological Network Inference (SEBINI), an interactive environment for the deployment and testing of network inference algorithms that use high-throughput data, and the Collective Analysis of Biological Interaction Networks (CABIN), software that allows integration and analysis of protein-protein interaction and gene-to-gene regulatory evidence obtained from multiple sources, to allow interactions computed by BEPro to be stored, visualized, and further analyzed. Incorporating BEPro into SEBINI and automatically feeding the resulting inferred network into CABIN, we have created a structured workflow for protein-protein network inference and supplemental analysis from sets of mass spectrometry bait-prey experiment data. SEBINI demo site: https://www.emsl.pnl.gov /SEBINI/ Contact: ronald.taylor@pnl.gov. BEPro is available at http://www.pnl.gov/statistics/BEPro3/index.htm. Contact: ds.daly@pnl.gov. CABIN is available at http://www.sysbio.org/dataresources/cabin.stm. Contact: mudita.singhal@pnl.gov.

  11. PathPPI: an integrated dataset of human pathways and protein-protein interactions.

    PubMed

    Tang, HaiLin; Zhong, Fan; Liu, Wei; He, FuChu; Xie, HongWei

    2015-06-01

    Integration of pathway and protein-protein interaction (PPI) data can provide more information that could lead to new biological insights. PPIs are usually represented by a simple binary model, whereas pathways are represented by more complicated models. We developed a series of rules for transforming protein interactions from pathway to binary model, and the protein interactions from seven pathway databases, including PID, BioCarta, Reactome, NetPath, INOH, SPIKE and KEGG, were transformed based on these rules. These pathway-derived binary protein interactions were integrated with PPIs from other five PPI databases including HPRD, IntAct, BioGRID, MINT and DIP, to develop integrated dataset (named PathPPI). More detailed interaction type and modification information on protein interactions can be preserved in PathPPI than other existing datasets. Comparison analysis results indicate that most of the interaction overlaps values (O AB) among these pathway databases were less than 5%, and these databases must be used conjunctively. The PathPPI data was provided at http://proteomeview.hupo.org.cn/PathPPI/PathPPI.html. PMID:25591449

  12. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  13. TOWARDS A PROBABILISTIC RECOGNITION CODE FOR PROTEIN-DNA INTERACTIONS

    SciTech Connect

    P. BENOS; ET AL

    2000-09-01

    We are investigating the rules that govern protein-DNA interactions, using a statistical mechanics based formalism that is related to the Boltzmann Machine of the neural net literature. Our approach is data-driven, in which probabilistic algorithms are used to model protein-DNA interactions, given SELEX and phage data as input. Under the ''one-to-one'' model for interactions (i.e. one amino acid contacts one base), we can successfully identify the wild-type binding sites of EGR and MIG protein families. The predictions using our method are the same or better than that of methods existing in the literature, however our methodology offers the potential to capitalize in quantitative detail on more data as it becomes available.

  14. Physiological functions of the TRPM4 channels via protein interactions

    PubMed Central

    Lee, Young-Sun; Kim, Eunju; Hwang, Eun Mi; Park, Jae-Yong

    2015-01-01

    Transient Receptor Potential, Melastatin-related, member 4 (TRPM4) channels are Ca2+-activated Ca2+-impermeable cation channels. These channels are expressed in various types of mammalian tissues including the brain and are implicated in many diverse physiological and pathophysiological conditions. In the past several years, the trafficking processes and regulatory mechanism of these channels and their interacting proteins have been uncovered. Here in this minireview, we summarize the current understanding of the trafficking mechanism of TRPM4 channels on the plasma membrane as well as heteromeric complex formation via protein interactions. We also describe physiological implications of protein-TRPM4 interactions and suggest TRPM4 channels as therapeutic targets in many related diseases. [BMB Reports 2015; 48(1): 1-5] PMID:25441424

  15. Glycoarray Technologies: Deciphering Interactions from Proteins to Live Cell Responses.

    PubMed

    Puvirajesinghe, Tania M; Turnbull, Jeremy E

    2016-01-01

    Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein-glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate-protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies (with a special focus on glycosaminoglycan applications) and their emerging applications to the study of complex interactions between glycans and whole living cells. PMID:27600069

  16. Preferential Interactions and the Effect of Protein PEGylation

    PubMed Central

    Holm, Louise Stenstrup; Thulstrup, Peter W.; Kasimova, Marina R.; van de Weert, Marco

    2015-01-01

    Background PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of protein drugs, whereas formulation excipients are used for stabilization of proteins during storage. Here we investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein. Methodology/Principal Findings The model protein hen egg white lysozyme was doubly PEGylated on two lysines with 5 kDa linear PEGs (mPEG-succinimidyl valerate, MW 5000) and studied in the absence and presence of preferentially excluded sucrose and preferentially bound guanine hydrochloride. Structural characterization by far- and near-UV circular dichroism spectroscopy was supplemented by investigation of protein thermal stability with the use of differential scanning calorimetry, far and near-UV circular dichroism and fluorescence spectroscopy. It was found that PEGylated lysozyme was stabilized by the preferentially excluded excipient and destabilized by the preferentially bound excipient in a similar manner as lysozyme. However, compared to lysozyme in all cases the melting transition was lower by up to a few degrees and the calorimetric melting enthalpy was decreased to half the value for PEGylated lysozyme. The ratio between calorimetric and van’t Hoff enthalpy suggests that our PEGylated lysozyme is a dimer. Conclusion/Significance The PEGylated model protein displayed similar stability responses to the addition of preferentially active excipients. This suggests that formulation principles using preferentially interacting excipients are similar for PEGylated and non-PEGylated proteins. PMID:26230338

  17. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  18. Dynamic imaging of protein-protein interactions by MP-FLIM

    NASA Astrophysics Data System (ADS)

    Ameer-Beg, Simon M.; Peter, Marion; Keppler, Melanie D.; Prag, Soren; Barber, Paul R.; Ng, Tony C.; Vojnovic, Borivoj

    2005-03-01

    The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

  19. Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

    PubMed

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  20. Detecting protein complexes in protein interaction networks using a ranking algorithm with a refined merging procedure

    PubMed Central

    2014-01-01

    Background Developing suitable methods for the identification of protein complexes remains an active research area. It is important since it allows better understanding of cellular functions as well as malfunctions and it consequently leads to producing more effective cures for diseases. In this context, various computational approaches were introduced to complement high-throughput experimental methods which typically involve large datasets, are expensive in terms of time and cost, and are usually subject to spurious interactions. Results In this paper, we propose ProRank+, a method which detects protein complexes in protein interaction networks. The presented approach is mainly based on a ranking algorithm which sorts proteins according to their importance in the interaction network, and a merging procedure which refines the detected complexes in terms of their protein members. ProRank + was compared to several state-of-the-art approaches in order to show its effectiveness. It was able to detect more protein complexes with higher quality scores. Conclusions The experimental results achieved by ProRank + show its ability to detect protein complexes in protein interaction networks. Eventually, the method could potentially identify previously-undiscovered protein complexes. The datasets and source codes are freely available for academic purposes at http://faculty.uaeu.ac.ae/nzaki/Research.htm. PMID:24944073

  1. The RNA Binding Motif Protein 15B (RBM15B/OTT3) Is a Functional Competitor of Serine-Arginine (SR) Proteins and Antagonizes the Positive Effect of the CDK11p110-Cyclin L2α Complex on Splicing*

    PubMed Central

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H.; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J.; Lahti, Jill M.

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11p110 binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11p110, cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11p110, cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  2. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

    PubMed

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J; Lahti, Jill M

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  3. [Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

    PubMed

    Beniaminov, A D; Krasnov, G S; Dmitriev, A A; Puzanov, G A; Snopok, B A; Senchenko, V N; Kashuba, V I

    2016-01-01

    Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation. PMID:27414789

  4. Proteins that interact with GTP during sporulation of Bacillus subtilis

    SciTech Connect

    Mitchell, C.; Vary, J.C. )

    1989-06-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link ({alpha}-{sup 32}P)GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either ({alpha}-{sup 32}P)GTP or ({gamma}-{sup 32}P)GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms.

  5. Reconstruction and Application of Protein–Protein Interaction Network

    PubMed Central

    Hao, Tong; Peng, Wei; Wang, Qian; Wang, Bin; Sun, Jinsheng

    2016-01-01

    The protein-protein interaction network (PIN) is a useful tool for systematic investigation of the complex biological activities in the cell. With the increasing interests on the proteome-wide interaction networks, PINs have been reconstructed for many species, including virus, bacteria, plants, animals, and humans. With the development of biological techniques, the reconstruction methods of PIN are further improved. PIN has gradually penetrated many fields in biological research. In this work we systematically reviewed the development of PIN in the past fifteen years, with respect to its reconstruction and application of function annotation, subsystem investigation, evolution analysis, hub protein analysis, and regulation mechanism analysis. Due to the significant role of PIN in the in-depth exploration of biological process mechanisms, PIN will be preferred by more and more researchers for the systematic study of the protein systems in various kinds of organisms. PMID:27338356

  6. Protein-protein interactions in intracellular Ca2+-release channel function.

    PubMed Central

    MacKrill, J J

    1999-01-01

    Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4, 5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein-protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein-protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are 'customized' by the accessory proteins present. PMID:9895277

  7. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  8. Protein-lipid interactions in bilayer membranes: A lattice model

    PubMed Central

    Pink, David A.; Chapman, Dennis

    1979-01-01

    A lattice model has been developed to study the effects of intrinsic membrane proteins upon the thermodynamic properties of a lipid bilayer membrane. We assume that only nearest-neighbor van der Waals and steric interactions are important and that the polar group interactions can be represented by effective pressure—area terms. Phase diagrams, the temperature T0, which locates the gel—fluid melting, the transition enthalpy, and correlations were calculated by mean field and cluster approximations. Average lipid chain areas and chain areas when the lipid is in a given protein environment were obtained. Proteins that have a “smooth” homogeneous surface (“cholesterol-like”) and those that have inhomogeneous surfaces or that bind lipids specifically were considered. We find that T0 can vary depending upon the interactions and that another peak can appear upon the shoulder of the main peak which reflects the melting of a eutectic mixture. The transition enthalpy decreases generally, as was found before, but when a second peak appears departures from this behavior reflect aspects of the eutectic mixture. We find that proteins have significant nonzero probabilities for being adjacent to one another so that no unbroken “annulus” of lipid necessarily exists around a protein. If T0 does not increase much, or decreases, with increasing c, then lipids adjacent to a protein cannot all be all-trans on the time scale (10-7 sec) of our system. Around a protein the lipid correlation depth is about one lipid layer, and this increases with c. Possible consequences of ignoring changes in polar group interactions due to clustering of proteins are discussed. PMID:286996

  9. Evaluation of two dependency parsers on biomedical corpus targeted at protein-protein interactions.

    PubMed

    Pyysalo, Sampo; Ginter, Filip; Pahikkala, Tapio; Boberg, Jorma; Järvinen, Jouni; Salakoski, Tapio

    2006-06-01

    We present an evaluation of Link Grammar and Connexor Machinese Syntax, two major broad-coverage dependency parsers, on a custom hand-annotated corpus consisting of sentences regarding protein-protein interactions. In the evaluation, we apply the notion of an interaction subgraph, which is the subgraph of a dependency graph expressing a protein-protein interaction. We measure the performance of the parsers for recovery of individual dependencies, fully correct parses, and interaction subgraphs. For Link Grammar, an open system that can be inspected in detail, we further perform a comprehensive failure analysis, report specific causes of error, and suggest potential modifications to the grammar. We find that both parsers perform worse on biomedical English than previously reported on general English. While Connexor Machinese Syntax significantly outperforms Link Grammar, the failure analysis suggests specific ways in which the latter could be modified for better performance in the domain. PMID:16099201

  10. Investigation of protein-protein interactions in living cells by chemical crosslinking and mass spectrometry.

    PubMed

    Sinz, Andrea

    2010-08-01

    The identification of protein-protein interactions within their physiological environment is the key to understanding biological processes at the molecular level. However, the artificial nature of in vitro experiments, with their lack of other cellular components, may obstruct observations of specific cellular processes. In vivo analyses can provide information on the processes within a cell that might not be observed in vitro. Chemical crosslinking combined with mass spectrometric analysis of the covalently connected binding partners allows us to identify interacting proteins and to map their interface regions directly in the cell. In this paper, different in vivo crosslinking strategies for deriving information on protein-protein interactions in their physiological environment are described. PMID:20076950

  11. Some remarks on prediction of protein-protein interaction with machine learning.

    PubMed

    Zhang, Shao-Wu; Wei, Ze-Gang

    2015-01-01

    Protein-protein interactions (PPIs) play a key role in many cellular processes. Uncovering the PPIs and their function within the cell is a challenge of post-genomic biology and will improve our understanding of disease and help in the development of novel methods for disease diagnosis and forensics. The experimental methods currently used to identify PPIs are both time-consuming and expensive, and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. These obstacles could be overcome by developing computational approaches to predict PPIs and validate the obtained experimental results. In this work, we will describe the recent advances in predicting protein-protein interaction from the following aspects: i) the benchmark dataset construction, ii) the sequence representation approaches, iii) the common machine learning algorithms, and iv) the cross-validation test methods and assessment metrics. PMID:25548927

  12. Influence of homology and node age on the growth of protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Bottinelli, Arianna; Bassetti, Bruno; Lagomarsino, Marco Cosentino; Gherardi, Marco

    2012-10-01

    Proteins participating in a protein-protein interaction network can be grouped into homology classes following their common ancestry. Proteins added to the network correspond to genes added to the classes, so the dynamics of the two objects are intrinsically linked. Here we first introduce a statistical model describing the joint growth of the network and the partitioning of nodes into classes, which is studied through a combined mean-field and simulation approach. We then employ this unified framework to address the specific issue of the age dependence of protein interactions through the definition of three different node wiring or divergence schemes. A comparison with empirical data indicates that an age-dependent divergence move is necessary in order to reproduce the basic topological observables together with the age correlation between interacting nodes visible in empirical data. We also discuss the possibility of nontrivial joint partition and topology observables.

  13. Scoring functions for prediction of protein-ligand interactions.

    PubMed

    Wang, Jui-Chih; Lin, Jung-Hsin

    2013-01-01

    The scoring functions for protein-ligand interactions plays central roles in computational drug design, virtual screening of chemical libraries for new lead identification, and prediction of possible binding targets of small chemical molecules. An ideal scoring function for protein-ligand interactions is expected to be able to recognize the native binding pose of a ligand on the protein surface among decoy poses, and to accurately predict the binding affinity (or binding free energy) so that the active molecules can be discriminated from the non-active ones. Due to the empirical nature of most, if not all, scoring functions for protein-ligand interactions, the general applicability of empirical scoring functions, especially to domains far outside training sets, is a major concern. In this review article, we will explore the foundations of different classes of scoring functions, their possible limitations, and their suitable application domains. We also provide assessments of several scoring functions on weakly-interacting protein-ligand complexes, which will be useful information in computational fragment-based drug design or virtual screening. PMID:23016847

  14. Nonlinear dynamics of specific DNA-protein interactions

    NASA Astrophysics Data System (ADS)

    Dwiputra, D.; Hidayat, W.; Khairani, R.; Zen, F. P.

    2016-03-01

    Interactions between DNA binding protein and specific base pairs of nucleic acid is critical for biological process. We propose a new model of DNA-protein interactions to depict the dynamics of specific DNA-protein interactions. Hydrogen bonds (H-bonds) are, among the other intermolecular interactions in DNA, the most distinctive in term of specificity of molecular bonds. As H-bonds account for specificity, we only consider the dynamics affected by H-bonds between DNA base pairs and H-bonds connecting protein side chains and DNA. The H-bonds are modelled by Morse potentials and coupling terms in the Hamiltonian of coupled oscillators resembling a coupling between planar DNA chain and a protein molecule. In this paper we give a perturbative approach as an attempt for a soliton solution. The solution is in the form of nonlinear travelling wave having the amplitudes satisfying coupled nonlinear Schrodinger equations and is interpreted as the mediator for nonlocal transmittance of biological information in DNA.

  15. Functional Studies of DNA-Protein Interactions Using FRET Techniques.

    PubMed

    Blouin, Simon; Craggs, Timothy D; Lafontaine, Daniel A; Penedo, J Carlos

    2015-01-01

    Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of protein-DNA interactions. PMID:26404147

  16. Interaction of receptor-activity-modifying protein1 with tubulin.

    PubMed

    Kunz, Thomas H; Mueller-Steiner, Sarah; Schwerdtfeger, Kerstin; Kleinert, Peter; Troxler, Heinz; Kelm, Jens M; Ittner, Lars M; Fischer, Jan A; Born, Walter

    2007-08-01

    Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors. PMID:17493758

  17. Interactions of polyphenols with carbohydrates, lipids and proteins.

    PubMed

    Jakobek, Lidija

    2015-05-15

    Polyphenols are secondary metabolites in plants, investigated intensively because of their potential positive effects on human health. Their bioavailability and mechanism of positive effects have been studied, in vitro and in vivo. Lately, a high number of studies takes into account the interactions of polyphenols with compounds present in foods, like carbohydrates, proteins or lipids, because these food constituents can have significant effects on the activity of phenolic compounds. This paper reviews the interactions between phenolic compounds and lipids, carbohydrates and proteins and their impact on polyphenol activity. PMID:25577120

  18. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  19. Discovering pathways by orienting edges in protein interaction networks

    PubMed Central

    Gitter, Anthony; Klein-Seetharaman, Judith; Gupta, Anupam; Bar-Joseph, Ziv

    2011-01-01

    Modern experimental technology enables the identification of the sensory proteins that interact with the cells’ environment or various pathogens. Expression and knockdown studies can determine the downstream effects of these interactions. However, when attempting to reconstruct the signaling networks and pathways between these sources and targets, one faces a substantial challenge. Although pathways are directed, high-throughput protein interaction data are undirected. In order to utilize the available data, we need methods that can orient protein interaction edges and discover high-confidence pathways that explain the observed experimental outcomes. We formalize the orientation problem in weighted protein interaction graphs as an optimization problem and present three approximation algorithms based on either weighted Boolean satisfiability solvers or probabilistic assignments. We use these algorithms to identify pathways in yeast. Our approach recovers twice as many known signaling cascades as a recent unoriented signaling pathway prediction technique and over 13 times as many as an existing network orientation algorithm. The discovered paths match several known signaling pathways and suggest new mechanisms that are not currently present in signaling databases. For some pathways, including the pheromone signaling pathway and the high-osmolarity glycerol pathway, our method suggests interesting and novel components that extend current annotations. PMID:21109539

  20. Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins

    PubMed Central

    Holthusen, Kirsten; Talaty, Pooja

    2015-01-01

    ABSTRACT Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton. IMPORTANCE LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins. PMID:25948738

  1. Atom depth analysis delineates mechanisms of protein intermolecular interactions

    SciTech Connect

    Alocci, Davide; Bernini, Andrea; Niccolai, Neri

    2013-07-12

    Highlights: •3D atom depth analysis is proposed to identify different layers in protein structures. •Amino acid contents for each layers have been analyzed for a large protein dataset. •Charged amino acids in the most external layer are present at very different extents. •Atom depth indexes of K residues reflect their side chains flexibility. •Mobile surface charges can be responsible for long range protein–protein recognition. -- Abstract: The systematic analysis of amino acid distribution, performed inside a large set of resolved protein structures, sheds light on possible mechanisms driving non random protein–protein approaches. Protein Data Bank entries have been selected using as filters a series of restrictions ensuring that the shape of protein surface is not modified by interactions with large or small ligands. 3D atom depth has been evaluated for all the atoms of the 2,410 selected structures. The amino acid relative population in each of the structural layers formed by grouping atoms on the basis of their calculated depths, has been evaluated. We have identified seven structural layers, the inner ones reproducing the core of proteins and the outer one incorporating their most protruding moieties. Quantitative analysis of amino acid contents of structural layers identified, as expected, different behaviors. Atoms of Q, R, K, N, D residues are increasingly more abundant in going from core to surfaces. An opposite trend is observed for V, I, L, A, C, and G. An intermediate behavior is exhibited by P, S, T, M, W, H, F and Y. The outer structural layer hosts predominantly E and K residues whose charged moieties, protruding from outer regions of the protein surface, reorient free from steric hindrances, determining specific electrodynamics maps. This feature may represent a protein signature for long distance effects, driving the formation of encounter complexes and the eventual short distance approaches that are required for protein–protein

  2. Graphlet-based edge clustering reveals pathogen-interacting proteins

    PubMed Central

    Solava, R. W.; Michaels, R. P.; Milenković, T.

    2012-01-01

    Motivation: Prediction of protein function from protein interaction networks has received attention in the post-genomic era. A popular strategy has been to cluster the network into functionally coherent groups of proteins and assign the entire cluster with a function based on functions of its annotated members. Traditionally, network research has focused on clustering of nodes. However, clustering of edges may be preferred: nodes belong to multiple functional groups, but clustering of nodes typically cannot capture the group overlap, while clustering of edges can. Clustering of adjacent edges that share many neighbors was proposed recently, outperforming different node clustering methods. However, since some biological processes can have characteristic ‘signatures’ throughout the network, not just locally, it may be of interest to consider edges that are not necessarily adjacent. Results: We design a sensitive measure of the ‘topological similarity’ of edges that can deal with edges that are not necessarily adjacent. We cluster edges that are similar according to our measure in different baker's yeast protein interaction networks, outperforming existing node and edge clustering approaches. We apply our approach to the human network to predict new pathogen-interacting proteins. This is important, since these proteins represent drug target candidates. Availability: Software executables are freely available upon request. Contact: tmilenko@nd.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22962470

  3. Protein-protein interaction networks: unraveling the wiring of molecular machines within the cell.

    PubMed

    De Las Rivas, Javier; Fontanillo, Celia

    2012-11-01

    Mapping and understanding of the protein interaction networks with their key modules and hubs can provide deeper insights into the molecular machinery underlying complex phenotypes. In this article, we present the basic characteristics and definitions of protein networks, starting with a distinction of the different types of associations between proteins. We focus the review on protein-protein interactions (PPIs), a subset of associations defined as physical contacts between proteins that occur by selective molecular docking in a particular biological context. We present such definition as opposed to other types of protein associations derived from regulatory, genetic, structural or functional relations. To determine PPIs, a variety of binary and co-complex methods exist; however, not all the technologies provide the same information and data quality. A way of increasing confidence in a given protein interaction is to integrate orthogonal experimental evidences. The use of several complementary methods testing each single interaction assesses the accuracy of PPI data and tries to minimize the occurrence of false interactions. Following this approach there have been important efforts to unify primary databases of experimentally proven PPIs into integrated databases. These meta-databases provide a measure of the confidence of interactions based on the number of experimental proofs that report them. As a conclusion, we can state that integrated information allows the building of more reliable interaction networks. Identification of communities, cliques, modules and hubs by analysing the topological parameters and graph properties of the protein networks allows the discovery of central/critical nodes, which are candidates to regulate cellular flux and dynamics. PMID:22908212

  4. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectromet