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Sample records for helicase interacting protein

  1. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    Warner, Dennis R.; Bhattacherjee, Vasker; Yin, Xiaolong; Singh, Saurabh; Mukhopadhyay, Partha; Pisano, M. Michele; Greene, Robert M.

    2004-01-01

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  2. Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

    Awate, Sanket; Brosh, Robert M

    2017-06-08

    Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

  3. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

    Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

    2007-01-01

    RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

  4. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  5. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  6. GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

    Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

    2016-12-01

    In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

    Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

    2002-06-28

    Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

  8. ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

    Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

    2017-01-01

    Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

    Hyun, Moonjung; Park, Sojin; Kim, Eunsun; Kim, Do-Hyung; Lee, Se-Jin; Koo, Hyeon-Sook; Seo, Yeon-Soo; Ahn, Byungchan

    2012-02-21

    The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

  10. Helicase properties of the Escherichia coli UvrAb protein complex

    Oh, E.Y.; Grossman, L.

    1987-01-01

    The Escherichia coli UvrA protein has an associated ATPase activity with a turnover number affected by the presence of UvrB protein as well as by DNA. Specifically, the structure of DNA significantly influences the turnover rate of the UvrAB ATPase activity. Double-stranded DNA maximally activates the turnover rate 10-fold whereas single-stranded DNA maximally activates the turnover rate 20-fold, suggesting that the mode of interaction of UvrAB protein with different DNAs is distinctive. We have previously shown that the UvrAB protein complex, driven by the binding energy of ATP, can locally unwind supercoiled DNA. The nature of the DNA unwinding activity and single-stranded DNA activation of ATPase activity suggest potential helicase activity. In the presence of a number of helicase substrates, the UvrAB complex, indeed, manifests a strand-displacement activity-unwinding short duplexes and D-loop DNA, thereby generating component DNA structures. The energy for the activity is derived from ATP or dATP hydrolysis. Unlike the E. coli DnaB, the UvrAB helicase is sensitive to UV-induced photoproducts

  11. The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

    Copsey, Alice C; Cooper, Simon; Parker, Robert; Lineham, Ella; Lapworth, Cuzack; Jallad, Deema; Sweet, Steve; Morley, Simon J

    2017-08-30

    DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading. © 2017 The Author(s).

  12. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  13. Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

    Yoomi Choi

    Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

  14. Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins

    Rebecca Bish

    2015-07-01

    Full Text Available DDX6 (p54/RCK is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58 of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2 and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2. We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6’s multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6’s interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions

  15. The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5

    Abdullah Shah

    2017-01-01

    Full Text Available DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m6A RNA demethylase. The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.

  16. SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

    Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

    2011-01-01

    In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

  17. Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

    Suhasini, Avvaru N; Rawtani, Nina A; Wu, Yuliang

    2011-01-01

    Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact...

  18. Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

    Bhushan, Lokesh; Abraham, Ambily; Choudhury, Nirupam Roy; Rana, Vipin Singh; Mukherjee, Sunil Kumar; Savithri, Handanahal Subbarao

    2015-04-01

    The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

  19. Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

    Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

    2015-01-01

    DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

  20. RECQ HELICASE RECQL4 PARTICIPATES IN NON-HOMOLOGOUS END JOINING AND INTERACTS WITH THE KU COMPLEX

    Shamanna, Raghavendra A; Singh, Dharmendra Kumar; Lu, Huiming

    2014-01-01

    -irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-dependent protein kinase (DNA-PK) complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding...... of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase...

  1. Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function

    Miller, Justin M.; Enemark, Eric J.

    2015-01-01

    In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. PMID:26539061

  2. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  3. Crystal structure of Middle East respiratory syndrome coronavirus helicase.

    Wei Hao

    2017-06-01

    Full Text Available Middle East respiratory syndrome coronavirus (MERS-CoV remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

  4. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  5. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-04-29

    Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  6. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    Zhang, Yuan; Palla, Mirkó; Liao, Jung-Chi; Sun, Andrew

    2013-01-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116. (paper)

  7. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  8. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

    2011-04-20

    XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

  9. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Close encounters for the first time: Helicase interactions with DNA damage.

    Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

    2015-09-01

    DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

  11. The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1

    Nagahama, Masami; Yamazoe, Takeshi; Hara, Yoshimitsu; Tani, Katsuko; Tsuji, Akihiko; Tagaya, Mitsuo

    2006-01-01

    Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase, whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone

  12. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity

    Schwendener, S.; Raynard, S.; Paliwal, S.; Cheng, A.; Kanagaraj, R.; Shevelev, Igor; Stark, J.M.; Sung, P.; Janscak, P.

    2010-01-01

    Roč. 285, č. 21 (2010), s. 15739-15745 ISSN 0021-9258 Grant - others:NIH(US) R01CA120954; NIH(US) ES015632; SNSF(CH) 3100A0-116008 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA helicase * double-strand breaks * homologous recombination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.328, year: 2010

  14. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  15. Bloom syndrome helicase in meiosis: Pro-crossover functions of an anti-crossover protein.

    Hatkevich, Talia; Sekelsky, Jeff

    2017-09-01

    The functions of the Bloom syndrome helicase (BLM) and its orthologs are well characterized in mitotic DNA damage repair, but their roles within the context of meiotic recombination are less clear. In meiotic recombination, multiple repair pathways are used to repair meiotic DSBs, and current studies suggest that BLM may regulate the use of these pathways. Based on literature from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, we present a unified model for a critical meiotic role of BLM and its orthologs. In this model, BLM and its orthologs utilize helicase activity to regulate the use of various pathways in meiotic recombination by continuously disassembling recombination intermediates. This unwinding activity provides the meiotic program with a steady pool of early recombination substrates, increasing the probability for a DSB to be processed by the appropriate pathway. As a result of BLM activity, crossovers are properly placed throughout the genome, promoting proper chromosomal disjunction at the end of meiosis. This unified model can be used to further refine the complex role of BLM and its orthologs in meiotic recombination. © 2017 WILEY Periodicals, Inc.

  16. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth

    2014-01-01

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers

  17. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  18. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

    2002-01-01

    Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

  19. Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

    Matsuo, Eiko [Microbiology and Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-City 657-8501 (Japan); Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom); Leon, Esther; Matthews, Steve J. [Division of Molecular Biosciences, Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ (United Kingdom); Roy, Polly, E-mail: polly.roy@lshtm.ac.uk [Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom)

    2014-09-05

    Highlights: • NMR analysis on BTV VP6 reveals two large loop regions. • The loss of a loop (aa 34–130) does not affect the overall fold of the protein. • A region of VP6 (aa 34–92) is not required for BTV replication. • A region of VP6 (aa 93–130) plays an essential role in the virus replication. - Abstract: Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

  20. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-01-01

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  1. Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization.

    Yen-Hua Huang

    Full Text Available The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204 showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196-228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205-228 is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein-protein interaction, not via the DNA-protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

  2. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

    Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

    2015-01-01

    Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

  3. Aquaporin Protein-Protein Interactions

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  4. Protein-Protein Interaction Databases

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  5. Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

    Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

    1990-02-25

    The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

  6. RecQL5 promotes genome stabilization through two parallel mechanisms--interacting with RNA polymerase II and acting as a helicase.

    Islam, M Nurul; Fox, David; Guo, Rong; Enomoto, Takemi; Wang, Weidong

    2010-05-01

    The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

  7. Targeting Dengue Virus NS-3 Helicase by Ligand based Pharmacophore Modeling and Structure based Virtual Screening

    Halim, Sobia A.; Khan, Shanza; Khan, Ajmal; Wadood, Abdul; Mabood, Fazal; Hussain, Javid; Al-Harrasi, Ahmed

    2017-10-01

    Dengue fever is an emerging public health concern, with several million viral infections occur annually, for which no effective therapy currently exist. Non-structural protein 3 (NS-3) Helicase encoded by the dengue virus (DENV) is considered as a potential drug target to design new and effective drugs against dengue. Helicase is involved in unwinding of dengue RNA. This study was conducted to design new NS-3 Helicase inhibitor by in silico ligand- and structure based approaches. Initially ligand-based pharmacophore model was generated that was used to screen a set of 1201474 compounds collected from ZINC Database. The compounds matched with the pharmacophore model were docked into the active site of NS-3 helicase. Based on docking scores and binding interactions, twenty five compounds are suggested to be potential inhibitors of NS3 Helicase. The pharmacokinetic properties of these hits were predicted. The selected hits revealed acceptable ADMET properties. This study identified potential inhibitors of NS-3 Helicase in silico, and can be helpful in the treatment of Dengue.

  8. Our interests in protein-protein interactions

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  9. Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors

    Saydam, Nurten; Kanagaraj, Radhakrishnan; Dietschy, Tobias

    2007-01-01

    is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSalpha), MSH2/MSH3 (MutSbeta) and MLH1/PMS2 (MutLalpha) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSalpha and MutSbeta can strongly...

  10. Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

    Heijden, van der M.W.; Carette, J.E.; Reinhoud, P.J.; Haegi, A.; Bol, J.F.

    2001-01-01

    Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and

  11. Interactive protein manipulation

    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.

  12. Interactive protein manipulation

    2003-01-01

    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. Binding of MCM-interacting proteins to ATP-binding site in MCM6

    Hosoi A

    2016-03-01

    Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

  14. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  16. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

  17. Overcoming natural replication barriers: differential helicase requirements.

    Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

    2012-02-01

    DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

  18. Evolution of protein-protein interactions

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  19. Purification and crystallization of Kokobera virus helicase

    De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno; Forrester, Naomi L.; Gould, Ernest; Canard, Bruno; Mattevi, Andrea

    2007-01-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å

  20. Helicase-dependent amplification of nucleic acids.

    Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

    2013-10-11

    Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

  1. Purification and crystallization of Kokobera virus helicase

    De Colibus, Luigi; Speroni, Silvia [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy); Coutard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Forrester, Naomi L.; Gould, Ernest [Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR (United Kingdom); Canard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Mattevi, Andrea, E-mail: mattevi@ipvgen.unipv.it [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy)

    2007-03-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.

  2. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    Peng Liu

    2015-01-01

    Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

  3. Molecular Dynamics of the ZIKA Virus NS3 Helicase

    Raubenolt, Bryan; Rick, Steven; The Rick Group Team

    The recent outbreaks of the ZIKA virus (ZIKV) and its connection to microcephaly in newborns has raised its awareness as a global threat and many scientific research efforts are currently underway in attempt to create a vaccine. Molecular Dynamics is a powerful method of investigating the physical behavior of protein complexes. ZIKV is comprised of 3 structural and 7 nonstructural proteins. The NS3 helicase protein appears to play a significant role in the replication complex and its inhibition could be a crucial source of antiviral drug design. This research primarily focuses on studying the structural dynamics, over the course of few hundred nanoseconds, of NS3 helicase in the free state, as well as in complex form with human ssRNA, ATP, and an analogue of GTP. RMSD and RMSF plots of each simulation will provide details on the forces involved in the overall stability of the active and inactive states. Furthermore, free energy calculations on a per residue level will reveal the most interactive residues between states and ultimately the primary driving force behind these interactions. Together these analyses will provide highly relevant information on the binding surface chemistry and thus serve as the basis for potential drug design.

  4. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally; Parish, Joanna L

    2017-01-01

    In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2 Y131A ) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2 Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2 WT ), the chromatin-bound pool of E2 Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2 Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2 WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2 Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection

  5. Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

    Atsushi Furuta

    2014-01-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3 helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3 and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

  6. Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

    Plötner, Björn; Nurmi, Markus; Fischer, Axel; Watanabe, Mutsumi; Schneeberger, Korbinian; Holm, Svante; Vaid, Neha; Schöttler, Mark Aurel; Walther, Dirk; Hoefgen, Rainer; Weigel, Detlef; Laitinen, Roosa A E

    2017-07-01

    Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F 2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  7. RecQ Helicases

    Larsen, Nicolai Balle; Hickson, Ian D

    2013-01-01

    The RecQ family of DNA helicases is highly conserved throughout -evolution, and is important for the maintenance of genome stability. In humans, five RecQ family members have been identified: BLM, WRN, RECQ4, RECQ1 and RECQ5. Defects in three of these give rise to Bloom's syndrome (BLM), Werner...

  8. Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2.

    Absmeier, Eva; Becke, Christian; Wollenhaupt, Jan; Santos, Karine F; Wahl, Markus C

    2017-01-02

    RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.

  9. Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Brisbarre, Nadège; Lamballerie, Xavier de; Coutard, Bruno; Canard, Bruno; Khromykh, Alexander; Bolognesi, Martino

    2006-01-01

    The C-terminal 440 amino acids of the NS3 protein from Kunjin virus (Flaviviridae) code for a helicase. The protein has been overexpressed and crystallized. Characterization of the isolated monoclinic crystal form and diffraction data (at 3.0 Å resolution) are presented, together with a preliminary molecular-replacement solution. Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3′ nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds

  10. Detecting mutually exclusive interactions in protein-protein interaction maps.

    Sá nchez Claros, Carmen; Tramontano, Anna

    2012-01-01

    Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  11. Detecting mutually exclusive interactions in protein-protein interaction maps.

    Sánchez Claros, Carmen

    2012-06-08

    Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  12. Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

    Fred E Indig

    Full Text Available The Werner protein (WRNp, a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL, an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA.These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

  13. Database of Interacting Proteins (DIP)

    U.S. Department of Health & Human Services — The DIP database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent...

  14. Velocity and processivity of helicase unwinding of double-stranded nucleic acids

    Betterton, M D; Juelicher, F

    2005-01-01

    Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed

  15. Scoring functions for protein-protein interactions.

    Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

    2013-12-01

    The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. A role for the fission yeast Rqh1 helicase in chromosome segregation

    Win, Thein Z; Mankouri, Hocine W; Hickson, Ian D

    2005-01-01

    Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled...

  17. Structural mechanisms of human RecQ helicases WRN and BLM

    Ken eKitano

    2014-10-01

    Full Text Available The RecQ family DNA helicases WRN (Werner syndrome protein and BLM (Bloom syndrome protein play a key role in protecting the genome against deleterious changes. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. WRN and BLM are distinguished from other helicases by possessing signature tandem domains toward the C terminus, referred to as the RecQ C-terminal (RQC and helicase-and-ribonuclease D-C-terminal (HRDC domains. Although the precise function of the HRDC domain remains unclear, the previous crystal structure of a WRN RQC-DNA complex visualized a central role for the RQC domain in recognizing, binding and unwinding DNA at branch points. In particular, a prominent hairpin structure (the β-wing within the RQC winged-helix motif acts as a scalpel to induce the unpairing of a Watson-Crick base pair at the DNA duplex terminus. A similar RQC-DNA interaction was also observed in the recent crystal structure of a BLM-DNA complex. I review the latest structures of WRN and BLM, and then provide a docking simulation of BLM with a Holliday junction. The model offers an explanation for the efficient branch migration activity of the RecQ family toward recombination and repair intermediates.

  18. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  19. Inhibition of RNA Helicases of ssRNA+ Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families

    Irene Briguglio

    2011-01-01

    Full Text Available Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

  20. Oligomeric protein structure networks: insights into protein-protein interactions

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  1. A new role for FBP21 as regulator of Brr2 helicase activity.

    Henning, Lisa M; Santos, Karine F; Sticht, Jana; Jehle, Stefanie; Lee, Chung-Tien; Wittwer, Malte; Urlaub, Henning; Stelzl, Ulrich; Wahl, Markus C; Freund, Christian

    2017-07-27

    Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Intercellular protein-protein interactions at synapses.

    Yang, Xiaofei; Hou, Dongmei; Jiang, Wei; Zhang, Chen

    2014-06-01

    Chemical synapses are asymmetric intercellular junctions through which neurons send nerve impulses to communicate with other neurons or excitable cells. The appropriate formation of synapses, both spatially and temporally, is essential for brain function and depends on the intercellular protein-protein interactions of cell adhesion molecules (CAMs) at synaptic clefts. The CAM proteins link pre- and post-synaptic sites, and play essential roles in promoting synapse formation and maturation, maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. Alteration of the interactions of CAMs leads to structural and functional impairments, which results in many neurological disorders, such as autism, Alzheimer's disease and schizophrenia. Therefore, it is crucial to understand the functions of CAMs during development and in the mature neural system, as well as in the pathogenesis of some neurological disorders. Here, we review the function of the major classes of CAMs, and how dysfunction of CAMs relates to several neurological disorders.

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

    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.

  4. Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

    Hood, Iris V.; Berger, James M.

    2016-05-31

    Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

  5. A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

    Nikolay Kovalev

    2012-02-01

    Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV. To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-RNA, rendering the RNA compatible for initiation of (+-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which is another host factor for TBSV, play non-overlapping functions to enhance (+-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV, a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

  6. Saccharomyces cerevisiae Hrq1 requires a long 3′-tailed DNA substrate for helicase activity

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-01-01

    Highlights: ► Hrq1 has intrinsic 3′–5′ helicase and DNA strand annealing activities. ► Hrq1 requires a long 3′-tail for efficient DNA unwinding. ► Helicase activity of Hrq1 is stimulated by a fork structure. ► Hrq1 is a moderately processive helicase. -- Abstract: RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3′–5′ helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3′-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases.

  7. Genome-wide identification of SF1 and SF2 helicases from archaea.

    Chamieh, Hala; Ibrahim, Hiba; Kozah, Juliana

    2016-01-15

    Archaea microorganisms have long been used as model organisms for the study of protein molecular machines. Archaeal proteins are particularly appealing to study since archaea, even though prokaryotic, possess eukaryotic-like cellular processes. Super Family I (SF1) and Super Family II (SF2) helicase families have been studied in many model organisms, little is known about their presence and distribution in archaea. We performed an exhaustive search of homologs of SF1 and SF2 helicase proteins in 95 complete archaeal genomes. In the present study, we identified the complete sets of SF1 and SF2 helicases in archaea. Comparative analysis between archaea, human and the bacteria E. coli SF1 and SF2 helicases, resulted in the identification of seven helicase families conserved among representatives of the domains of life. This analysis suggests that these helicase families are highly conserved throughout evolution. We highlight the conserved motifs of each family and characteristic domains of the detected families. Distribution of SF1/SF2 families show that Ski2-like, Lhr, Sfth and Rad3-like helicases are ubiquitous among archaeal genomes while the other families are specific to certain archaeal groups. We also report the presence of a novel SF2 helicase specific to archaea domain named Archaea Specific Helicase (ASH). Phylogenetic analysis indicated that ASH has evolved in Euryarchaeota and is evolutionary related to the Ski2-like family with specific characteristic domains. Our study provides the first exhaustive analysis of SF1 and SF2 helicases from archaea. It expands the variety of SF1 and SF2 archaeal helicases known to exist to date and provides a starting point for new biochemical and genetic studies needed to validate their biological functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Saccharomyces cerevisiae Hrq1 requires a long 3'-tailed DNA substrate for helicase activity.

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-10-26

    RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3'-5' helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3'-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    Ghosh, Sharmistha; Hamdan, Samir; Richardson, Charles C.

    2010-01-01

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    Ghosh, Sharmistha

    2010-04-06

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Specificity and affinity quantification of protein-protein interactions.

    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  12. Coevolution of interacting fertilization proteins.

    Nathaniel L Clark

    2009-07-01

    Full Text Available Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female-male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.

  13. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  14. A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

    Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

    2012-08-01

    Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

  15. X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

    Tortorici, M Alejandra; Duquerroy, Stéphane; Kwok, Jane; Vonrhein, Clemens; Perez, Javier; Lamp, Benjamin; Bricogne, Gerard; Rümenapf, Till; Vachette, Patrice; Rey, Félix A

    2015-04-01

    Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the

  16. Drosophila Protein interaction Map (DPiM)

    Guruharsha, K.G.; Obar, Robert A.; Mintseris, Julian; Aishwarya, K.; Krishnan, R.T.; VijayRaghavan, K.; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when,...

  17. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Furuta, Atsushi; Tsubuki, Masayoshi; Endoh, Miduki; Miyamoto, Tatsuki; Tanaka, Junichi; Abdus Salam, Kazi; Akimitsu, Nobuyoshi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2015-01-01

    Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition. PMID:26262613

  18. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Atsushi Furuta

    2015-08-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3 helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

  19. PBDE: Structure-Activity Studies for the Inhibition of Hepatitis C Virus NS3 Helicase

    Kazi Abdus Salam

    2014-04-01

    Full Text Available The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3 is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1 on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1 against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

  20. Coarse-grain modelling of protein-protein interactions

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  1. Mitochondrial helicases and mitochondrial genome maintenance

    de Souza-Pinto, Nadja C; Aamann, Maria Diget; Kulikowicz, Tomasz

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...

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

    Zheng Sun

    2014-01-01

    Full Text Available 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.

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

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

    2015-02-23

    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.

  4. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

    Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including pl...

  5. Can infrared spectroscopy provide information on protein-protein interactions?

    Haris, Parvez I

    2010-08-01

    For most biophysical techniques, characterization of protein-protein interactions is challenging; this is especially true with methods that rely on a physical phenomenon that is common to both of the interacting proteins. Thus, for example, in IR spectroscopy, the carbonyl vibration (1600-1700 cm(-1)) associated with the amide bonds from both of the interacting proteins will overlap extensively, making the interpretation of spectral changes very complicated. Isotope-edited infrared spectroscopy, where one of the interacting proteins is uniformly labelled with (13)C or (13)C,(15)N has been introduced as a solution to this problem, enabling the study of protein-protein interactions using IR spectroscopy. The large shift of the amide I band (approx. 45 cm(-1) towards lower frequency) upon (13)C labelling of one of the proteins reveals the amide I band of the unlabelled protein, enabling it to be used as a probe for monitoring conformational changes. With site-specific isotopic labelling, structural resolution at the level of individual amino acid residues can be achieved. Furthermore, the ability to record IR spectra of proteins in diverse environments means that isotope-edited IR spectroscopy can be used to structurally characterize difficult systems such as protein-protein complexes bound to membranes or large insoluble peptide/protein aggregates. In the present article, examples of application of isotope-edited IR spectroscopy for studying protein-protein interactions are provided.

  6. PSAIA – Protein Structure and Interaction Analyzer

    Vlahoviček Kristian

    2008-04-01

    Full Text Available Abstract Background PSAIA (Protein Structure and Interaction Analyzer was developed to compute geometric parameters for large sets of protein structures in order to predict and investigate protein-protein interaction sites. Results In addition to most relevant established algorithms, PSAIA offers a new method PIADA (Protein Interaction Atom Distance Algorithm for the determination of residue interaction pairs. We found that PIADA produced more satisfactory results than comparable algorithms implemented in PSAIA. Particular advantages of PSAIA include its capacity to combine different methods to detect the locations and types of interactions between residues and its ability, without any further automation steps, to handle large numbers of protein structures and complexes. Generally, the integration of a variety of methods enables PSAIA to offer easier automation of analysis and greater reliability of results. PSAIA can be used either via a graphical user interface or from the command-line. Results are generated in either tabular or XML format. Conclusion In a straightforward fashion and for large sets of protein structures, PSAIA enables the calculation of protein geometric parameters and the determination of location and type for protein-protein interaction sites. XML formatted output enables easy conversion of results to various formats suitable for statistic analysis. Results from smaller data sets demonstrated the influence of geometry on protein interaction sites. Comprehensive analysis of properties of large data sets lead to new information useful in the prediction of protein-protein interaction sites.

  7. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    Mukherjee, Sourav; Weiner, Warren S.; Schroeder, Chad E.; Simpson, Denise S.; Hanson, Alicia M.; Sweeney, Noreena L.; Marvin, Rachel K.; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J.; Frick, David N.

    2014-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previousl...

  8. Understanding Protein-Protein Interactions Using Local Structural Features

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

    2013-01-01

    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...... to score the likelihood of the interaction between two proteins and to develop a method for the prediction of PPIs. We have tested our method on several sets with unbalanced ratios of interactions and non-interactions to simulate real conditions, obtaining accuracies higher than 25% in the most unfavorable...

  9. Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

    Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

    2015-11-02

    The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

  10. Integral UBL domain proteins: a family of proteasome interacting proteins

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact...

  11. Evolutionary reprograming of protein-protein interaction specificity.

    Akiva, Eyal; Babbitt, Patricia C

    2015-10-22

    Using mutation libraries and deep sequencing, Aakre et al. study the evolution of protein-protein interactions using a toxin-antitoxin model. The results indicate probable trajectories via "intermediate" proteins that are promiscuous, thus avoiding transitions via non-interactions. These results extend observations about other biological interactions and enzyme evolution, suggesting broadly general principles. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. A conserved mammalian protein interaction network.

    Åsa Pérez-Bercoff

    Full Text Available Physical interactions between proteins mediate a variety of biological functions, including signal transduction, physical structuring of the cell and regulation. While extensive catalogs of such interactions are known from model organisms, their evolutionary histories are difficult to study given the lack of interaction data from phylogenetic outgroups. Using phylogenomic approaches, we infer a upper bound on the time of origin for a large set of human protein-protein interactions, showing that most such interactions appear relatively ancient, dating no later than the radiation of placental mammals. By analyzing paired alignments of orthologous and putatively interacting protein-coding genes from eight mammals, we find evidence for weak but significant co-evolution, as measured by relative selective constraint, between pairs of genes with interacting proteins. However, we find no strong evidence for shared instances of directional selection within an interacting pair. Finally, we use a network approach to show that the distribution of selective constraint across the protein interaction network is non-random, with a clear tendency for interacting proteins to share similar selective constraints. Collectively, the results suggest that, on the whole, protein interactions in mammals are under selective constraint, presumably due to their functional roles.

  13. HCVpro: Hepatitis C virus protein interaction database

    Kwofie, Samuel K.; Schaefer, Ulf; Sundararajan, Vijayaraghava Seshadri; Bajic, Vladimir B.; Christoffels, Alan G.

    2011-01-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers

  14. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita.

    Ballew, Bari J; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P; Savage, Sharon A

    2013-04-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.

  15. Protein-protein interactions and cancer: targeting the central dogma.

    Garner, Amanda L; Janda, Kim D

    2011-01-01

    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  16. Protein Annotation from Protein Interaction Networks and Gene Ontology

    Nguyen, Cao D.; Gardiner, Katheleen J.; Cios, Krzysztof J.

    2011-01-01

    We introduce a novel method for annotating protein function that combines Naïve Bayes and association rules, and takes advantage of the underlying topology in protein interaction networks and the structure of graphs in the Gene Ontology. We apply our method to proteins from the Human Protein Reference Database (HPRD) and show that, in comparison with other approaches, it predicts protein functions with significantly higher recall with no loss of precision. Specifically, it achieves 51% precis...

  17. Mass spectrometric analysis of protein interactions

    Borch, Jonas; Jørgensen, Thomas J. D.; Roepstorff, Peter

    2005-01-01

    Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now...... available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope...... labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably....

  18. Genome-Wide Analysis of the RNA Helicase Gene Family in Gossypium raimondii

    Jie Chen

    2014-03-01

    Full Text Available The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes, DEAH-box (52 genes, or DExD/H-box (58 genes in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5% are within the identified syntenic blocks. Sixty-six (40.99% helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

  19. Human cancer protein-protein interaction network: a structural perspective.

    Gozde Kar

    2009-12-01

    Full Text Available Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network. The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%. We illustrate the interface related affinity properties of two cancer-related hub

  20. Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

    Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

    2012-07-11

    Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

  1. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-05

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

  2. MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

    Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

    2014-01-01

    As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

  3. Yeast Interacting Proteins Database: YPR103W, YOR047C [Yeast Interacting Proteins Database

    Full Text Available tein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors...gulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf

  4. Molecular simulations of lipid-mediated protein-protein interactions

    de Meyer, F.J.M.; Venturoli, M.; Smit, B.

    2008-01-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the

  5. Yeast Interacting Proteins Database: YLR447C, YOR047C [Yeast Interacting Proteins Database

    Full Text Available xpression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Sp...; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; act

  6. Yeast Interacting Proteins Database: YGR013W, YKL012W [Yeast Interacting Proteins Database

    Full Text Available tion U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the formation of the second commitm... PRP40 U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the form...ation of the second commitment complex Rows with this prey as prey (1) Rows with

  7. Information assessment on predicting protein-protein interactions

    Gerstein Mark

    2004-10-01

    Full Text Available Abstract Background Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. Results Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. Conclusions In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the

  8. A Mesoscopic Model for Protein-Protein Interactions in Solution

    Lund, Mikael; Jönsson, Bo

    2003-01-01

    Protein self-association may be detrimental in biological systems, but can be utilized in a controlled fashion for protein crystallization. It is hence of considerable interest to understand how factors like solution conditions prevent or promote aggregation. Here we present a computational model describing interactions between protein molecules in solution. The calculations are based on a molecular description capturing the detailed structure of the protein molecule using x-ray or nuclear ma...

  9. Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

    Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

    2014-10-17

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

  10. Interaction between plate make and protein in protein crystallisation screening.

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  11. Mapping Protein-Protein Interactions by Quantitative Proteomics

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2010-01-01

    spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions.......Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...

  12. Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples

    Lachapelle, Sophie; Gagné, Jean-Philippe; Garand, Chantal

    2011-01-01

    Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale prot...

  13. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  14. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  15. Building blocks for protein interaction devices

    Grünberg, Raik; Ferrar, Tony S.; van der Sloot, Almer M.; Constante, Marco; Serrano, Luis

    2010-01-01

    Here, we propose a framework for the design of synthetic protein networks from modular protein–protein or protein–peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part–based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors controlling protein expression in Escherichia coli: obstruction of translation initiation by mRNA secondary structure or toxicity of individual domains. Eventually, 13 proteins were purified for further characterization. Starting from well-established biotechnological tools, two general–purpose interaction input and two readout devices were built and characterized in vitro. Constitutive interaction input was achieved with a pair of synthetic leucine zippers. The second interaction was drug-controlled utilizing the rapamycin-induced binding of FRB(T2098L) to FKBP12. The interaction kinetics of both devices were analyzed by surface plasmon resonance. Readout was based on Förster resonance energy transfer between fluorescent proteins and was quantified for various combinations of input and output devices. Our results demonstrate the feasibility of parts-based protein synthetic biology. Additionally, we identify future challenges and limitations of modular design along with approaches to address them. PMID:20215443

  16. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Selection of peptides interfering with protein-protein interaction.

    Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

    2009-01-01

    Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

  18. Data management of protein interaction networks

    Cannataro, Mario

    2012-01-01

    Interactomics: a complete survey from data generation to knowledge extraction With the increasing use of high-throughput experimental assays, more and more protein interaction databases are becoming available. As a result, computational analysis of protein-to-protein interaction (PPI) data and networks, now known as interactomics, has become an essential tool to determine functionally associated proteins. From wet lab technologies to data management to knowledge extraction, this timely book guides readers through the new science of interactomics, giving them the tools needed to: Generate

  19. Enterovirus Exposure Uniquely Discriminates Type 1 Diabetes Patients with a Homozygous from a Heterozygous Melanoma Differentiation-Associated Protein 5/Interferon Induced with Helicase C Domain 1 A946T Genotype.

    Schulte, Barbara M; Gielen, Paul R; Kers-Rebel, Esther D; Prosser, Amy C; Lind, Katharina; Flodström-Tullberg, Malin; Tack, Cees J; Elving, Lammy D; Adema, Gosse J

    2016-09-01

    In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5, predisposes to disease. We hypothesized that the strength of innate antienteroviral responses is affected in autoimmune type 1 diabetes patients and linked to the A946T polymorphism. We compared induction of interferon-stimulated genes (ISGs) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in healthy individuals and diabetes patients upon stimulation with enterovirus, enterovirus-antibody complexes, or ligands mimicking infection in relation to the A946T polymorphism. Overall, PBMCs of diabetes patients and healthy donors showed comparable ISG induction upon stimulation. No differences were observed in DCs. Interestingly, the data imply that the magnitude of responses to enterovirus and enterovirus-antibody complexes in PBMCs is critically influenced by the A946T polymorphism and elevated in heterozygotes compared to TT homozygous individuals in autoimmune diabetes patients, but not healthy controls. These data imply an intrinsic difference in the responses to enterovirus and enterovirus-antibody complexes in diabetes patients carrying a TT risk genotype compared to heterozygotes that may influence control of enterovirus clearance.

  20. Protein annotation from protein interaction networks and Gene Ontology.

    Nguyen, Cao D; Gardiner, Katheleen J; Cios, Krzysztof J

    2011-10-01

    We introduce a novel method for annotating protein function that combines Naïve Bayes and association rules, and takes advantage of the underlying topology in protein interaction networks and the structure of graphs in the Gene Ontology. We apply our method to proteins from the Human Protein Reference Database (HPRD) and show that, in comparison with other approaches, it predicts protein functions with significantly higher recall with no loss of precision. Specifically, it achieves 51% precision and 60% recall versus 45% and 26% for Majority and 24% and 61% for χ²-statistics, respectively. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk

    Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan

    2016-01-01

    BACKGROUND: BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants...

  2. On the role of electrostatics on protein-protein interactions

    Zhang, Zhe; Witham, Shawn; Alexov, Emil

    2011-01-01

    The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity. PMID:21572182

  3. Protein-Protein Interactions (PPI) reagents: | Office of Cancer Genomics

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below.

  4. Protein-Protein Interaction Reagents | Office of Cancer Genomics

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu

  5. Yeast Interacting Proteins Database: YGL237C, YOR047C [Yeast Interacting Proteins Database

    Full Text Available ene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding prote... expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein

  6. Yeast Interacting Proteins Database: YKL002W, YOR047C [Yeast Interacting Proteins Database

    Full Text Available ene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding prote...xpression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Sp

  7. Non-interacting surface solvation and dynamics in protein-protein interactions

    Visscher, Koen M.; Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

    2015-01-01

    Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo

  8. Water-Protein Interactions: The Secret of Protein Dynamics

    Silvia Martini

    2013-01-01

    Full Text Available Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R1NS and selective (R1SE spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells.

  9. Interactions between whey proteins and kaolinite surfaces

    Barral, S.; Villa-Garcia, M.A.; Rendueles, M.; Diaz, M.

    2008-01-01

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered

  10. Interactions between whey proteins and kaolinite surfaces

    Barral, S. [Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Villa-Garcia, M.A. [Department of Organic and Inorganic Chemistry, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)], E-mail: mavg@uniovi.es; Rendueles, M. [Project Management Area, University of Oviedo, Independencia 13, 33004 Oviedo (Spain); Diaz, M. [Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)

    2008-07-15

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered.

  11. HCVpro: Hepatitis C virus protein interaction database

    Kwofie, Samuel K.

    2011-12-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers. In furtherance of these goals, we have developed the hepatitis C virus protein interaction database (HCVpro) by integrating manually verified hepatitis C virus-virus and virus-human protein interactions curated from literature and databases. HCVpro is a comprehensive and integrated HCV-specific knowledgebase housing consolidated information on PPIs, functional genomics and molecular data obtained from a variety of virus databases (VirHostNet, VirusMint, HCVdb and euHCVdb), and from BIND and other relevant biology repositories. HCVpro is further populated with information on hepatocellular carcinoma (HCC) related genes that are mapped onto their encoded cellular proteins. Incorporated proteins have been mapped onto Gene Ontologies, canonical pathways, Online Mendelian Inheritance in Man (OMIM) and extensively cross-referenced to other essential annotations. The database is enriched with exhaustive reviews on structure and functions of HCV proteins, current state of drug and vaccine development and links to recommended journal articles. Users can query the database using specific protein identifiers (IDs), chromosomal locations of a gene, interaction detection methods, indexed PubMed sources as well as HCVpro, BIND and VirusMint IDs. The use of HCVpro is free and the resource can be accessed via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. © 2011 Elsevier B.V.

  12. Cdt1 stabilizes an open MCM ring for helicase loading.

    Frigola, Jordi; He, Jun; Kinkelin, Kerstin; Pye, Valerie E; Renault, Ludovic; Douglas, Max E; Remus, Dirk; Cherepanov, Peter; Costa, Alessandro; Diffley, John F X

    2017-06-23

    ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.

  13. The nuclear import of RNA helicase A is mediated by importin-α3

    Aratani, Satoko; Oishi, Takayuki; Fujita, Hidetoshi; Nakazawa, Minako; Fujii, Ryouji; Imamoto, Naoko; Yoneda, Yoshihiro; Fukamizu, Akiyoshi; Nakajima, Toshihiro

    2006-01-01

    RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-α/β-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-α. Furthermore, the nuclear import of RHA-NLS was supported by importin-α1 and preferentially importin-α3. Our results indicate that the nuclear import of RHA is mediated by the importin-α3/importin-β-dependent pathway and suggest that the specificity for importin may regulate the functions of cargo proteins

  14. Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

    Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

    2014-01-01

    Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

  15. Inferring domain-domain interactions from protein-protein interactions with formal concept analysis.

    Susan Khor

    Full Text Available 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.

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

    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

  17. Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

    Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

    2011-05-20

    Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

  18. Detecting protein-protein interactions in living cells

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

  19. Yeast Interacting Proteins Database: YGL145W, YNL258C [Yeast Interacting Proteins Database

    Full Text Available ripheral membrane protein required for Golgi-to-ER retrograde traffic; component ... membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interact

  20. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  1. Analysis of Protein-Membrane Interactions

    Kemmer, Gerdi Christine

    Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions....... Discovered interactions were then probed on the level of the membrane using liposome-based assays. In the second part, a transmembrane protein was investigated. Assays to probe activity of the plasma membrane ATPase (Arabidopsis thaliana H+ -ATPase isoform 2 (AHA2)) in single liposomes using both giant...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...

  2. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: Implications for gene panel testing

    D.F. Easton (Douglas F.); F. Lesueur (Fabienne); B. Decker (Brennan); K. Michailidou (Kyriaki); J. Li (Jun); J. Allen (Jamie); C. Luccarini (Craig); K.A. Pooley (Karen); M. Shah (Mitul); M.K. Bolla (Manjeet); Q. Wang (Qin); J. Dennis (Joe); J. Ahmad (Jamil); E.R. Thompson (Ella); F. Damiola (Francesca); M. Pertesi (Maroulio); C. Voegele (Catherine); N. Mebirouk (Noura); N. Robinot (Nivonirina); G. Durand (Geoffroy); N. Forey (Nathalie); R.N. Luben (Robert); S. Ahmed (Shahana); K. Aittomäki (Kristiina); H. Anton-Culver (Hoda); V. Arndt (Volker); C. Baynes (Caroline); M.W. Beckmann (Matthias); J. Benítez (Javier); D. Van Den Berg (David); W.J. Blot (William); N.V. Bogdanova (Natalia); S.E. Bojesen (Stig); H. Brenner (Hermann); J. Chang-Claude (Jenny); K.S. Chia (Kee Seng); J.-Y. Choi (Ji-Yeob); D. Conroy (Don); A. Cox (Angela); S.S. Cross (Simon S.); K. Czene (Kamila); H. Darabi (Hatef); P. Devilee (Peter); M. Eriksson (Mats); P.A. Fasching (Peter); J.D. Figueroa (Jonine); H. Flyger (Henrik); F. Fostira (Florentia); M. García-Closas (Montserrat); G.G. Giles (Graham); G. Glendon (Gord); A. González-Neira (Anna); P. Guénel (Pascal); C.A. Haiman (Christopher A.); P. Hall (Per); S.N. Hart (Steven N.); J.M. Hartman (Joost); M.J. Hooning (Maartje); C.-N. Hsiung (Chia-Ni); H. Ito (Hidemi); A. Jakubowska (Anna); P.A. James (Paul A.); E.M. John (Esther M.); N. Johnson (Nichola); M. Jones (Michael); M. Kabisch (Maria); D. Kang (Daehee); V-M. Kosma (Veli-Matti); V. Kristensen (Vessela); D. Lambrechts (Diether); N. Li (Na); E. Myöhänen (Eija); H. Kemiläinen (Helena); A. Lindblom (Annika); J. Long (Jirong); A. Lophatananon (Artitaya); J. Lubinski (Jan); A. Mannermaa (Arto); K. Matsuo (Keitaro); S. Margolin (Sara); K. Matsuo (Keitaro); A. Meindl (Alfons); G. Mitchell (Gillian); K.R. Muir (K.); I. Nevelsteen (Ines); A.M.W. van den Ouweland (Ans); P. Peterlongo (Paolo); S.-Y. Phuah (Sze-Yee); K. Pykäs (Katri); S.M. Rowley (Simone M.); C-Y. Shen (Chen-Yang); R.K. Schmutzler (Rita); C.-Y. Shen (Chen-Yang); X.-O. Shu (Xiao-Ou); M.C. Southey (Melissa); H. Surowy (Harald); A.J. Swerdlow (Anthony ); S.-H. Teo (Soo-Hwang); R.A.E.M. Tollenaar (Rob); I.P. Tomlinson (Ian); C. Vachon (Celine); T. Truong (Thérèse); C. Vachon (Celine); S. Verhoef; M. Wong-Brown (Michelle); W. Zheng (Wei); Y. Zheng (Ying); H. Nevanlinna (Heli); R.J. Scott (Rodney); I.L. Andrulis (Irene); F.J. Couch (Fergus); J.L. Hopper (John); B. Burwinkel (Barbara); R. Winqvist (Robert); B. Burwinkel (Barbara); E.J. Sawyer (Elinor); M.K. Schmidt (Marjanka); A. Rudolph (Anja); T. Dörk (Thilo); S.L. Neuhausen (Susan); U. Hamann (Ute); S.L. Neuhausen (Susan L.); R.L. Milne (Roger); O. Fletcher (Olivia); P.D.P. Pharoah (Paul); I. Campbell (Ian); D. Goldgar (David); S.V. Tavtigian (Sean); D.E. Goldgar (David E.); S.V. Tavtigian (Sean V.); G. Chenevix-Trench (Georgia); K. Kleivi (Kristine); L. Ottestad (Lars); R. Karesen (Rolf); A. Langerød (Anita); E. Schlichting (Ellen); M.M. Holmen (Marit Muri); T. Sauer (Toril); V. Haakensen (Vilde); O. Engebråten (Olav); B. Naume (Bjorn); C.E. Kiserud (Cecile E.); K.V. Reinertsen (Kristin V.); Å. Helland (Åslaug); M. Riis (Margit); I. Bukholm (Ida); P.E. Lønning (Per ); S. Nord (Silje); G.G. Alnæs (Grethe)

    2016-01-01

    textabstractBackground BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein

  3. NMR Studies of Protein Hydration and Protein-Ligand Interactions

    Chong, Yuan

    Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

  4. Inferring high-confidence human protein-protein interactions

    Yu Xueping

    2012-05-01

    Full Text Available Abstract 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

  5. Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

    Sitaraman, Kalavathy; Chatterjee, Deb K

    2011-01-01

    In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

  6. Protein-protein interactions within late pre-40S ribosomes.

    Melody G Campbell

    2011-01-01

    Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

  7. A rice kinase-protein interaction map.

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

    2009-03-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

  8. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  9. Yeast Interacting Proteins Database: YOR047C, YKL038W [Yeast Interacting Proteins Database

    Full Text Available racts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a...Bait description Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose senso...rs Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator of the tra

  10. Yeast Interacting Proteins Database: YFR049W, YOR047C [Yeast Interacting Proteins Database

    Full Text Available protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator... (0) YOR047C STD1 Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sens...ors Snf3p and Rgt2p, and TATA-binding protein Spt15p; ac

  11. Deciphering peculiar protein-protein interacting modules in Deinococcus radiodurans

    Barkallah Insaf

    2009-04-01

    Full Text Available Abstract Interactomes of proteins under positive selection from ionizing-radiation-resistant bacteria (IRRB might be a part of the answer to the question as to how IRRB, particularly Deinococcus radiodurans R1 (Deira, resist ionizing radiation. Here, using the Database of Interacting Proteins (DIP and the Protein Structural Interactome (PSI-base server for PSI map, we have predicted novel interactions of orthologs of the 58 proteins under positive selection in Deira and other IRRB, but which are absent in IRSB. Among these, 18 domains and their interactomes have been identified in DNA checkpoint and repair; kinases pathways; energy and nucleotide metabolisms were the important biological processes that were found to be involved. This finding provides new clues to the cellular pathways that can to be important for ionizing-radiation resistance in Deira.

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

    Feng, Mei [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Kang, Hongsuk; Luan, Binquan [Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Yang, Zaixing [Institute of Quantitative Biology and Medicine, SRMP and RAD-X, and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 (China); Zhou, Ruhong, E-mail: ruhong@us.ibm.com [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Department of Chemistry, Columbia University, New York, New York 10027 (United States)

    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.

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

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

    2016-01-01

    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.

  14. Specificity of molecular interactions in transient protein-protein interaction interfaces.

    Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

    2006-11-15

    In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of

  15. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  16. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

    Ana Lúcia Leitão

    2015-01-01

    Full Text Available The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

  17. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

    Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  18. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

    2016-04-22

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  19. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  20. Targeting protein-protein interactions for parasite control.

    Christina M Taylor

    2011-04-01

    Full Text Available 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 orthologous 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

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

    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.

  2. PIWI Proteins and PIWI-Interacting RNA

    Han, Yi Neng; Li, Yuan; Xia, Sheng Qiang

    2017-01-01

    tissue types as well and play important roles in transposon silencing, epigenetic regulation, gene and protein regulation, genome rearrangement, spermatogenesis and germ stem-cell maintenance. PIWI proteins were first discovered in Drosophila and they play roles in spermatogenesis, germline stem-cell......P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNAs (ncRNAs) and interact with PIWI proteins. piRNAs were primarily described in the germline, but emerging evidence revealed that piRNAs are expressed in a tissue-specific manner among multiple human somatic...... maintenance, self-renewal, retrotransposons silencing and the male germline mobility control. A growing number of studies have demonstrated that several piRNA and PIWI proteins are aberrantly expressed in various kinds of cancers and may probably serve as a novel biomarker and therapeutic target for cancer...

  3. Yeast Interacting Proteins Database: YMR280C, YOR047C [Yeast Interacting Proteins Database

    Full Text Available olved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensor... glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, an

  4. Receptor-interacting protein (RIP) kinase family

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  5. Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

    Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

    2001-01-01

    The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

  6. Yeast Interacting Proteins Database: YOR358W, YOR047C [Yeast Interacting Proteins Database

    Full Text Available ; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; act...rotein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator o

  7. Yeast Interacting Proteins Database: YGL127C, YOR047C [Yeast Interacting Proteins Database

    Full Text Available ith protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regula...rotein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors

  8. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  9. Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

    He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

    2009-12-31

    We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

  10. A human homolog of the yeast nucleotide excision repair gene MMS19 interacts with transcription repair factor TFIIH through the XPB and XPD helicases.

    T. Seroz; G.S. Winkler (Sebastiaan); J. Auriol; R.A. Verhage; W. Vermeulen (Wim); B. Smit (Bep); J. Brouwer (Jaap); A.P.M. Eker (André); G. Weeda (Geert); J-M. Egly (Jean-Marc); J.H.J. Hoeijmakers (Jan)

    2000-01-01

    textabstractNucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro

  11. Identification of NAD interacting residues in proteins

    Raghava Gajendra PS

    2010-03-01

    Full Text Available Abstract Background Small molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs from amino acid sequence using bioinformatics tools. Results We extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC 0.47 with accuracy 74.13% at window length 17

  12. Enterovirus Exposure Uniquely Discriminates Type 1 Diabetes Patients with a Homozygous from a Heterozygous Melanoma Differentiation-Associated Protein 5/Interferon Induced with Helicase C Domain 1 A946T Genotype

    Schulte, B.M.; Gielen, P.R.; Kers-Rebel, E.D.; Prosser, A.C.; Lind, K.; Flodstrom-Tullberg, M.; Tack, C.J.J.; Elving, L.D.; Adema, G.J.

    2016-01-01

    In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma

  13. Next-Generation Sequencing for Binary Protein-Protein Interactions

    Bernhard eSuter

    2015-12-01

    Full Text Available The yeast two-hybrid (Y2H system exploits host cell genetics in order to display binary protein-protein interactions (PPIs via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS, and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  14. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  15. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  16. PCNA Structure and Interactions with Partner Proteins

    Oke, Muse; Zaher, Manal S.; Hamdan, Samir

    2018-01-01

    Proliferating cell nuclear antigen (PCNA) consists of three identical monomers that topologically encircle double-stranded DNA. PCNA stimulates the processivity of DNA polymerase δ and, to a less extent, the intrinsically highly processive DNA polymerase ε. It also functions as a platform that recruits and coordinates the activities of a large number of DNA processing proteins. Emerging structural and biochemical studies suggest that the nature of PCNA-partner proteins interactions is complex. A hydrophobic groove at the front side of PCNA serves as a primary docking site for the consensus PIP box motifs present in many PCNA-binding partners. Sequences that immediately flank the PIP box motif or regions that are distant from it could also interact with the hydrophobic groove and other regions of PCNA. Posttranslational modifications on the backside of PCNA could add another dimension to its interaction with partner proteins. An encounter of PCNA with different DNA structures might also be involved in coordinating its interactions. Finally, the ability of PCNA to bind up to three proteins while topologically linked to DNA suggests that it would be a versatile toolbox in many different DNA processing reactions.

  17. PCNA Structure and Interactions with Partner Proteins

    Oke, Muse

    2018-01-29

    Proliferating cell nuclear antigen (PCNA) consists of three identical monomers that topologically encircle double-stranded DNA. PCNA stimulates the processivity of DNA polymerase δ and, to a less extent, the intrinsically highly processive DNA polymerase ε. It also functions as a platform that recruits and coordinates the activities of a large number of DNA processing proteins. Emerging structural and biochemical studies suggest that the nature of PCNA-partner proteins interactions is complex. A hydrophobic groove at the front side of PCNA serves as a primary docking site for the consensus PIP box motifs present in many PCNA-binding partners. Sequences that immediately flank the PIP box motif or regions that are distant from it could also interact with the hydrophobic groove and other regions of PCNA. Posttranslational modifications on the backside of PCNA could add another dimension to its interaction with partner proteins. An encounter of PCNA with different DNA structures might also be involved in coordinating its interactions. Finally, the ability of PCNA to bind up to three proteins while topologically linked to DNA suggests that it would be a versatile toolbox in many different DNA processing reactions.

  18. Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions.

    Guruharsha, K G; Obar, Robert A; Mintseris, Julian; Aishwarya, K; Krishnan, R T; Vijayraghavan, K; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

  19. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    Haidar, Malak; de Laté , Perle Latré ; Kennedy, Eileen J.; Langsley, Gordon

    2017-01-01

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way

  20. Protein-protein interaction site predictions with three-dimensional probability distributions of interacting atoms on protein surfaces.

    Ching-Tai Chen

    Full Text Available Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins and were tested on an independent dataset (consisting of 142 proteins. The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted

  1. Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

  2. Tools for controlling protein interactions with light

    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

  3. Feature generation and representations for protein-protein interaction classification.

    Lan, Man; Tan, Chew Lim; Su, Jian

    2009-10-01

    Automatic detecting protein-protein interaction (PPI) relevant articles is a crucial step for large-scale biological database curation. The previous work adopted POS tagging, shallow parsing and sentence splitting techniques, but they achieved worse performance than the simple bag-of-words representation. In this paper, we generated and investigated multiple types of feature representations in order to further improve the performance of PPI text classification task. Besides the traditional domain-independent bag-of-words approach and the term weighting methods, we also explored other domain-dependent features, i.e. protein-protein interaction trigger keywords, protein named entities and the advanced ways of incorporating Natural Language Processing (NLP) output. The integration of these multiple features has been evaluated on the BioCreAtIvE II corpus. The experimental results showed that both the advanced way of using NLP output and the integration of bag-of-words and NLP output improved the performance of text classification. Specifically, in comparison with the best performance achieved in the BioCreAtIvE II IAS, the feature-level and classifier-level integration of multiple features improved the performance of classification 2.71% and 3.95%, respectively.

  4. [Interaction of protein with charged colloidal particles].

    Durdenko, E V; Kuznetsova, S M; Basova, L V; Tikhonenko, S A; Saburova, E A

    2011-01-01

    The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of alpha-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.

  5. Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

    Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

    2016-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of

  6. Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study

    Ito, Emma; Sahri, Daniela; Knippers, Rolf; Carstens, Eric B.

    2004-01-01

    IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells

  7. Topology-function conservation in protein-protein interaction networks.

    Davis, Darren; Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Stojmirovic, Aleksandar; Pržulj, Nataša

    2015-05-15

    Proteins underlay the functioning of a cell and the wiring of proteins in protein-protein interaction network (PIN) relates to their biological functions. Proteins with similar wiring in the PIN (topology around them) have been shown to have similar functions. This property has been successfully exploited for predicting protein functions. Topological similarity is also used to guide network alignment algorithms that find similarly wired proteins between PINs of different species; these similarities are used to transfer annotation across PINs, e.g. from model organisms to human. To refine these functional predictions and annotation transfers, we need to gain insight into the variability of the topology-function relationships. For example, a function may be significantly associated with specific topologies, while another function may be weakly associated with several different topologies. Also, the topology-function relationships may differ between different species. To improve our understanding of topology-function relationships and of their conservation among species, we develop a statistical framework that is built upon canonical correlation analysis. Using the graphlet degrees to represent the wiring around proteins in PINs and gene ontology (GO) annotations to describe their functions, our framework: (i) characterizes statistically significant topology-function relationships in a given species, and (ii) uncovers the functions that have conserved topology in PINs of different species, which we term topologically orthologous functions. We apply our framework to PINs of yeast and human, identifying seven biological process and two cellular component GO terms to be topologically orthologous for the two organisms. © The Author 2015. Published by Oxford University Press.

  8. Comparative structural analysis of human DEAD-box RNA helicases.

    Patrick Schütz

    2010-09-01

    Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  9. Comparative structural analysis of human DEAD-box RNA helicases.

    Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

    2010-09-30

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  10. Yeast Interacting Proteins Database: YOR302W, YOR047C [Yeast Interacting Proteins Database

    Full Text Available rol of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt...tein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt1

  11. Yeast Interacting Proteins Database: YNL258C, YKR022C [Yeast Interacting Proteins Database

    Full Text Available YNL258C DSL1 Peripheral membrane protein required for Golgi-to-ER retrograde traffi...equired for Golgi-to-ER retrograde traffic; component of the ER target site that interacts with coatomer, th...it ORF YNL258C Bait gene name DSL1 Bait description Peripheral membrane protein r

  12. Poly(ethylene glycol) interactions with proteins

    Hašek, Jindřich

    2006-01-01

    Roč. 2, č. 23 (2006), s. 613-618 ISSN 0044-2968. [European Powder Diffraction Conference /9./. Prague, 02.09.2004-05.09.2004] R&D Projects: GA ČR(CZ) GA204/02/0843 Institutional research plan: CEZ:AV0Z40500505 Keywords : poly(ethylene glycol) * PEO * protein-polymer interaction Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.897, year: 2006

  13. Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

  14. Notable Aspects of Glycan-Protein Interactions

    Miriam Cohen

    2015-09-01

    Full Text Available 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.

  15. Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

    Kusumoto-Matsuo, Rika; Opresko, Patricia L; Ramsden, Dale

    2010-01-01

    Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere...... D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo....

  16. DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

    Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr [Department of Life Sciences, University of Ulsan, Ulsan (Korea, Republic of); Bohr, Vilhelm A. [Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, Baltimore, MD (United States)

    2011-08-12

    Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

  17. Yeast Interacting Proteins Database: YDR176W, YDL239C [Yeast Interacting Proteins Database

    Full Text Available a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle pole...ining structure at the leading edge of the prospore membrane via interaction with spindle pole body componen...DY3 Prey description Protein required for spore wall formation, thought to mediate assembly of a Don1p-conta

  18. Detection of protein complex from protein-protein interaction network using Markov clustering

    Ochieng, P J; Kusuma, W A; Haryanto, T

    2017-01-01

    Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks. (paper)

  19. Quantifying the molecular origins of opposite solvent effects on protein-protein interactions.

    Vincent Vagenende

    Full Text Available Although the nature of solvent-protein interactions is generally weak and non-specific, addition of cosolvents such as denaturants and osmolytes strengthens protein-protein interactions for some proteins, whereas it weakens protein-protein interactions for others. This is exemplified by the puzzling observation that addition of glycerol oppositely affects the association constants of two antibodies, D1.3 and D44.1, with lysozyme. To resolve this conundrum, we develop a methodology based on the thermodynamic principles of preferential interaction theory and the quantitative characterization of local protein solvation from molecular dynamics simulations. We find that changes of preferential solvent interactions at the protein-protein interface quantitatively account for the opposite effects of glycerol on the antibody-antigen association constants. Detailed characterization of local protein solvation in the free and associated protein states reveals how opposite solvent effects on protein-protein interactions depend on the extent of dewetting of the protein-protein contact region and on structural changes that alter cooperative solvent-protein interactions at the periphery of the protein-protein interface. These results demonstrate the direct relationship between macroscopic solvent effects on protein-protein interactions and atom-scale solvent-protein interactions, and establish a general methodology for predicting and understanding solvent effects on protein-protein interactions in diverse biological environments.

  20. Protein interaction networks by proteome peptide scanning.

    Christiane Landgraf

    2004-01-01

    Full Text Available A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment, that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are

  1. A Small Molecule Inhibitor of the BLM Helicase Modulates Chromosome Stability in Human Cells

    Nguyen, Giang Huong; Dexheimer, Thomas S; Rosenthal, Andrew S

    2013-01-01

    The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM f...

  2. TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

    G.S. Winkler (Sebastiaan); U. Fiedler; W. Vermeulen (Wim); F. Coin (Frédéric); R.D. Wood (Richard); H.T.M. Timmers (Marc); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S.J. Araú jo; J-M. Egly (Jean-Marc)

    2000-01-01

    textabstractTFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and

  3. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

    Stefanie C Wolski

    2008-06-01

    Full Text Available DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

  4. Parallel force assay for protein-protein interactions.

    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.

  5. Parallel force assay for protein-protein interactions.

    Daniela Aschenbrenner

    Full Text Available 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.

  6. Structural view of the helicase reveals that Zika virus uses a conserved mechanism for unwinding RNA.

    Li, Lei; Wang, Jin; Jia, Zhihui; Shaw, Neil

    2018-04-01

    Recent studies suggest a link between infection by Zika virus (ZIKV) and the development of neurological complications. The lack of ZIKV-specific therapeutics has alarmed healthcare professionals worldwide. Here, crystal structures of apo and AMPPNP- and Mn 2+ -bound forms of the essential helicase of ZIKV refined to 1.78 and 1.3 Å resolution, respectively, are reported. The structures reveal a conserved trimodular topology of the helicase. ATP and Mn 2+ are tethered between two RecA-like domains by conserved hydrogen-bonding interactions. The binding of ligands induces the movement of backbone Cα and side-chain atoms. Numerous solvent molecules are observed in the vicinity of the AMPPNP, suggesting a role in catalysis. These high-resolution structures could be useful for the design of inhibitors targeting the helicase of ZIKV for the treatment of infections caused by ZIKV.

  7. Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry

    Liu, Fan; Heck, Albert J R

    2015-01-01

    Proteins are involved in almost all processes of the living cell. They are organized through extensive networks of interaction, by tightly bound macromolecular assemblies or more transiently via signaling nodes. Therefore, revealing the architecture of protein complexes and protein interaction

  8. Evaluation of clustering algorithms for protein-protein interaction networks

    van Helden Jacques

    2006-11-01

    Full Text Available Abstract Background Protein interactions are crucial components of all cellular processes. Recently, high-throughput methods have been developed to obtain a global description of the interactome (the whole network of protein interactions for a given organism. In 2002, the yeast interactome was estimated to contain up to 80,000 potential interactions. This estimate is based on the integration of data sets obtained by various methods (mass spectrometry, two-hybrid methods, genetic studies. High-throughput methods are known, however, to yield a non-negligible rate of false positives, and to miss a fraction of existing interactions. The interactome can be represented as a graph where nodes correspond with proteins and edges with pairwise interactions. In recent years clustering methods have been developed and applied in order to extract relevant modules from such graphs. These algorithms require the specification of parameters that may drastically affect the results. In this paper we present a comparative assessment of four algorithms: Markov Clustering (MCL, Restricted Neighborhood Search Clustering (RNSC, Super Paramagnetic Clustering (SPC, and Molecular Complex Detection (MCODE. Results A test graph was built on the basis of 220 complexes annotated in the MIPS database. To evaluate the robustness to false positives and false negatives, we derived 41 altered graphs by randomly removing edges from or adding edges to the test graph in various proportions. Each clustering algorithm was applied to these graphs with various parameter settings, and the clusters were compared with the annotated complexes. We analyzed the sensitivity of the algorithms to the parameters and determined their optimal parameter values. We also evaluated their robustness to alterations of the test graph. We then applied the four algorithms to six graphs obtained from high-throughput experiments and compared the resulting clusters with the annotated complexes. Conclusion This

  9. Rationalizing the chemical space of protein-protein interaction inhibitors.

    Sperandio, Olivier; Reynès, Christelle H; Camproux, Anne-Claude; Villoutreix, Bruno O

    2010-03-01

    Protein-protein interactions (PPIs) are one of the next major classes of therapeutic targets, although they are too intricate to tackle with standard approaches. This is due, in part, to the inadequacy of today's chemical libraries. However, the emergence of a growing number of experimentally validated inhibitors of PPIs (i-PPIs) allows drug designers to use chemoinformatics and machine learning technologies to unravel the nature of the chemical space covered by the reported compounds. Key characteristics of i-PPIs can then be revealed and highlight the importance of specific shapes and/or aromatic bonds, enabling the design of i-PPI-enriched focused libraries and, therefore, of cost-effective screening strategies. 2009 Elsevier Ltd. All rights reserved.

  10. The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

    Landry, Aaron P; Ding, Huangen

    2014-01-01

    Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

  11. The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

    Aaron P. Landry

    2014-01-01

    Full Text Available Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0. The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss and double-stranded (ds DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

  12. Protein complex prediction based on k-connected subgraphs in protein interaction network

    Habibi, Mahnaz; Eslahchi, Changiz; Wong, Limsoon

    2010-01-01

    Abstract Background Protein complexes play an important role in cellular mechanisms. Recently, several methods have been presented to predict protein complexes in a protein interaction network. In these methods, a protein complex is predicted as a dense subgraph of protein interactions. However, interactions data are incomplete and a protein complex does not have to be a complete or dense subgraph. Results We propose a more appropriate protein complex prediction method, CFA, that is based on ...

  13. Protein function prediction using neighbor relativity in protein-protein interaction network.

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Receptor-interacting protein (RIP) kinase family

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  15. A Novel Approach for Protein-Named Entity Recognition and Protein-Protein Interaction Extraction

    Meijing Li

    2015-01-01

    Full Text Available Many researchers focus on developing protein-named entity recognition (Protein-NER or PPI extraction systems. However, the studies about these two topics cannot be merged well; then existing PPI extraction systems’ Protein-NER still needs to improve. In this paper, we developed the protein-protein interaction extraction system named PPIMiner based on Support Vector Machine (SVM and parsing tree. PPIMiner consists of three main models: natural language processing (NLP model, Protein-NER model, and PPI discovery model. The Protein-NER model, which is named ProNER, identifies the protein names based on two methods: dictionary-based method and machine learning-based method. ProNER is capable of identifying more proteins than dictionary-based Protein-NER model in other existing systems. The final discovered PPIs extracted via PPI discovery model are represented in detail because we showed the protein interaction types and the occurrence frequency through two different methods. In the experiments, the result shows that the performances achieved by our ProNER and PPI discovery model are better than other existing tools. PPIMiner applied this protein-named entity recognition approach and parsing tree based PPI extraction method to improve the performance of PPI extraction. We also provide an easy-to-use interface to access PPIs database and an online system for PPIs extraction and Protein-NER.

  16. Fragment molecular orbital method for studying lanthanide interactions with proteins

    Tsushima, Satoru [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Komeiji, Y. [National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba (Japan); Mochizuki, Y. [Rikkyo Univ., Tokyo (Japan)

    2017-06-01

    The binding affinity of the calcium-binding protein calmodulin towards Eu{sup 3+} was studied as a model for lanthanide protein interactions in the large family of ''EF-hand'' calcium-binding proteins.

  17. Protein-Protein Interaction Network and Gene Ontology

    Choi, Yunkyu; Kim, Seok; Yi, Gwan-Su; Park, Jinah

    Evolution of computer technologies makes it possible to access a large amount and various kinds of biological data via internet such as DNA sequences, proteomics data and information discovered about them. It is expected that the combination of various data could help researchers find further knowledge about them. Roles of a visualization system are to invoke human abilities to integrate information and to recognize certain patterns in the data. Thus, when the various kinds of data are examined and analyzed manually, an effective visualization system is an essential part. One instance of these integrated visualizations can be combination of protein-protein interaction (PPI) data and Gene Ontology (GO) which could help enhance the analysis of PPI network. We introduce a simple but comprehensive visualization system that integrates GO and PPI data where GO and PPI graphs are visualized side-by-side and supports quick reference functions between them. Furthermore, the proposed system provides several interactive visualization methods for efficiently analyzing the PPI network and GO directedacyclic- graph such as context-based browsing and common ancestors finding.

  18. Unified Alignment of Protein-Protein Interaction Networks.

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

    2017-04-19

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

  19. Protein-protein interaction network-based detection of functionally similar proteins within species.

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

  20. Yeast Interacting Proteins Database: YDL239C, YDR273W [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...it as prey (1) YDR273W DON1 Meiosis-specific component of the spindle pole body, part of the leading... edge protein (LEP) coat, forms a ring-like structure at the leading edge of the prospore...ption Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading...description Meiosis-specific component of the spindle pole body, part of the leading edge protein (LEP) coat

  1. Protein-lipid interactions at interfaces

    Wilde, P.

    2000-04-01

    Full Text Available Foams and emulsions are both types of multiphase foods and are a dispersion of one immiscible phase (e.g. air or oil in another (e.g. water. Amphiphilic molecules (either proteins or chemical compounds are able to stabilise the interface between these phases and are termed emulsifiers. The ability of protein emulsifiers to bind lipid is reviewed, and the mechanisms underlying the behaviour of these and low molecular weight surfactants (LMWS at the interface are summarised. New research, exploiting atomic force microscopy, has given fresh insights into the mechanisms by which proteins and LMWS interact when both are present at the interface, compromising the stability of foams and emulsions stabilised by these mixtures. The understanding of component interactions at the interfacial level is essential if advances are to be made in the control and manipulation of multiphase foods during production and storage.Las espumas y las emulsiones son dispersiones de una fase inmiscible (ejemplo aire o aceite en otra (ejemplo agua. Las moléculas anfifílicas (bien proteínas o compuestos químicos pueden estabilizar la interfase y se denominan emulsionantes. En este artículo se revisa la habilidad de los emulsionantes proteínicos para enlazar lípidos y los mecanismos que subyacen en el comportamiento de estas moléculas así como de los tensioactivos de bajo peso molecular en la interfase. Recientes investigaciones que usan la microscopía han ofrecido visiones nuevas de los mecanismos mediante los cuales las proteínas y los tensioactivos de bajo peso molecular interaccionan cuando ambos están presentes en la interfase, comprometiendo la estabilidad de espumas y emulsiones estabilizadas por estas mezclas. El entendimiento de las interacciones entre componentes a nivel interfacial es esencial para lograr avances en el control y manipulación de alimentos multifases durante la producción y el almacenamiento.

  2. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

    John L Goodier

    Full Text Available MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC, inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1, Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.

  3. Interaction between actinides and protein: the calmodulin

    Brulfert, Florian

    2016-01-01

    Considering the environmental impact of the Fukushima nuclear accident, it is fundamental to study the mechanisms governing the effects of the released radionuclides on the biosphere and thus identify the molecular processes generating the transport and deposition of actinides, such as neptunium and uranium. However, the information about the microscopic aspect of the interaction between actinides and biological molecules (peptides, proteins...) is scarce. The data being mostly reported from a physiological point of view, the structure of the coordination sites remains largely unknown. These microscopic data are indeed essential for the understanding of the interdependency between structural aspect, function and affinity.The Calmodulin (CaM) (abbreviation for Calcium-Modulated protein), also known for its affinity towards actinides, acts as a metabolic regulator of calcium. This protein is a Ca carrier, which is present ubiquitously in the human body, may also bind other metals such as actinides. Thus, in case of a contamination, actinides that bind to CaM could avoid the protein to perform properly and lead to repercussions on a large range of vital functions.The complexation of Np and U was studied by EXAFS spectroscopy which showed that actinides were incorporated in a calcium coordination site. Once the thermodynamical and structural aspects studied, the impact of the coordination site distortion on the biological efficiency was analyzed. In order to evaluate these consequences, a calorimetric method based on enzyme kinetics was developed. This experiment, which was conducted with both uranium (50 - 500 nM) and neptunium (30 - 250 nM) showed a decrease of the heat produced by the enzymatic reaction with an increasing concentration of actinides in the medium. Our findings showed that the Calmodulin actinide complex works as an enzymatic inhibitor. Furthermore, at higher neptunium (250 nM) and uranium (500 nM) concentration the metals seem to have a poison

  4. Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

    Rossi, Marie L; Ghosh, Avik K; Kulikowicz, Tomasz

    2010-01-01

    Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer...... provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N...... activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases....

  5. A scored human protein-protein interaction network to catalyze genomic interpretation

    Li, Taibo; Wernersson, Rasmus; Hansen, Rasmus B

    2017-01-01

    Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap,......Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (In...

  6. Yeast Interacting Proteins Database: YDL239C, YLR423C [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...cription Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading

  7. Yeast Interacting Proteins Database: YDL239C, YPL070W [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...cription Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading

  8. Yeast Interacting Proteins Database: YDL239C, YML042W [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...iption Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading

  9. Yeast Interacting Proteins Database: YDL239C, YKL103C [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...ait description Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading

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

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

    2011-01-01

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

  11. Protein complex prediction in large ontology attributed protein-protein interaction networks.

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng; Xu, Bo

    2013-01-01

    Protein complexes are important for unraveling the secrets of cellular organization and function. Many computational approaches have been developed to predict protein complexes in protein-protein interaction (PPI) networks. However, most existing approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology (GO) annotation information. In this paper, we constructed ontology attributed PPI networks with PPI data and GO resource. After constructing ontology attributed networks, we proposed a novel approach called CSO (clustering based on network structure and ontology attribute similarity). Structural information and GO attribute information are complementary in ontology attributed networks. CSO can effectively take advantage of the correlation between frequent GO annotation sets and the dense subgraph for protein complex prediction. Our proposed CSO approach was applied to four different yeast PPI data sets and predicted many well-known protein complexes. The experimental results showed that CSO was valuable in predicting protein complexes and achieved state-of-the-art performance.

  12. The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

    Simona Cugusi

    2016-01-01

    Full Text Available RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi and microRNA (miRNA pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

  13. Molecular determinants of nucleolar translocation of RNA helicase A

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  14. Interaction of Proteins Identified in Human Thyroid Cells

    Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

    2013-01-01

    Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

  15. Interaction of Proteins Identified in Human Thyroid Cells

    Jessica Pietsch

    2013-01-01

    Full Text Available Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.

  16. Topology and weights in a protein domain interaction network--a novel way to predict protein interactions.

    Wuchty, Stefan

    2006-05-23

    While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions

  17. Topology and weights in a protein domain interaction network – a novel way to predict protein interactions

    Wuchty Stefan

    2006-05-01

    Full Text Available Abstract Background While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. Results We consider a web of interactions between protein domains of the Protein Family database (PFAM, which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Conclusion Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we

  18. The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients

    Jensen, Martin Borch; Dunn, Christopher A; Keijzers, Guido

    2012-01-01

    RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations....... These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase...

  19. Evidence of probabilistic behaviour in protein interaction networks

    Reifman Jaques

    2008-01-01

    Full Text Available Abstract Background Data from high-throughput experiments of protein-protein interactions are commonly used to probe the nature of biological organization and extract functional relationships between sets of proteins. What has not been appreciated is that the underlying mechanisms involved in assembling these networks may exhibit considerable probabilistic behaviour. Results We find that the probability of an interaction between two proteins is generally proportional to the numerical product of their individual interacting partners, or degrees. The degree-weighted behaviour is manifested throughout the protein-protein interaction networks studied here, except for the high-degree, or hub, interaction areas. However, we find that the probabilities of interaction between the hubs are still high. Further evidence is provided by path length analyses, which show that these hubs are separated by very few links. Conclusion The results suggest that protein-protein interaction networks incorporate probabilistic elements that lead to scale-rich hierarchical architectures. These observations seem to be at odds with a biologically-guided organization. One interpretation of the findings is that we are witnessing the ability of proteins to indiscriminately bind rather than the protein-protein interactions that are actually utilized by the cell in biological processes. Therefore, the topological study of a degree-weighted network requires a more refined methodology to extract biological information about pathways, modules, or other inferred relationships among proteins.

  20. RAIN: RNA-protein Association and Interaction Networks

    Junge, Alexander; Refsgaard, Jan Christian; Garde, Christian

    2017-01-01

    is challenging due to data heterogeneity. Here, we present a database of ncRNA-RNA and ncRNA-protein interactions and its integration with the STRING database of protein-protein interactions. These ncRNA associations cover four organisms and have been established from curated examples, experimental data...

  1. Annotating the protein-RNA interaction sites in proteins using evolutionary information and protein backbone structure.

    Li, Tao; Li, Qian-Zhong

    2012-11-07

    RNA-protein interactions play important roles in various biological processes. The precise detection of RNA-protein interaction sites is very important for understanding essential biological processes and annotating the function of the proteins. In this study, based on various features from amino acid sequence and structure, including evolutionary information, solvent accessible surface area and torsion angles (φ, ψ) in the backbone structure of the polypeptide chain, a computational method for predicting RNA-binding sites in proteins is proposed. When the method is applied to predict RNA-binding sites in three datasets: RBP86 containing 86 protein chains, RBP107 containing 107 proteins chains and RBP109 containing 109 proteins chains, better sensitivities and specificities are obtained compared to previously published methods in five-fold cross-validation tests. In order to make further examination for the efficiency of our method, the RBP107 dataset is used as training set, RBP86 and RBP109 datasets are used as the independent test sets. In addition, as examples of our prediction, RNA-binding sites in a few proteins are presented. The annotated results are consistent with the PDB annotation. These results show that our method is useful for annotating RNA binding sites of novel proteins.

  2. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  3. Predicting and validating protein interactions using network structure.

    Pao-Yang Chen

    2008-07-01

    Full Text Available Protein interactions play a vital part in the function of a cell. As experimental techniques for detection and validation of protein interactions are time consuming, there is a need for computational methods for this task. Protein interactions appear to form a network with a relatively high degree of local clustering. In this paper we exploit this clustering by suggesting a score based on triplets of observed protein interactions. The score utilises both protein characteristics and network properties. Our score based on triplets is shown to complement existing techniques for predicting protein interactions, outperforming them on data sets which display a high degree of clustering. The predicted interactions score highly against test measures for accuracy. Compared to a similar score derived from pairwise interactions only, the triplet score displays higher sensitivity and specificity. By looking at specific examples, we show how an experimental set of interactions can be enriched and validated. As part of this work we also examine the effect of different prior databases upon the accuracy of prediction and find that the interactions from the same kingdom give better results than from across kingdoms, suggesting that there may be fundamental differences between the networks. These results all emphasize that network structure is important and helps in the accurate prediction of protein interactions. The protein interaction data set and the program used in our analysis, and a list of predictions and validations, are available at http://www.stats.ox.ac.uk/bioinfo/resources/PredictingInteractions.

  4. Analysis of protein-protein interaction networks by means of annotated graph mining algorithms

    Rahmani, Hossein

    2012-01-01

    This thesis discusses solutions to several open problems in Protein-Protein Interaction (PPI) networks with the aid of Knowledge Discovery. PPI networks are usually represented as undirected graphs, with nodes corresponding to proteins and edges representing interactions among protein pairs. A large

  5. Regulation of PCNA-protein interactions for genome stability

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...

  6. Full Data of Yeast Interacting Proteins Database (Original Version) - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Full Text Available List Contact us Yeast Interacting Proteins Database Full Data of Yeast Interacting Proteins Database (Origin...al Version) Data detail Data name Full Data of Yeast Interacting Proteins Database (Original Version) DOI 10....18908/lsdba.nbdc00742-004 Description of data contents The entire data in the Yeast Interacting Proteins Database...eir interactions are required. Several sources including YPD (Yeast Proteome Database, Costanzo, M. C., Hoga...ematic name in the SGD (Saccharomyces Genome Database; http://www.yeastgenome.org /). Bait gene name The gen

  7. Gap junctions and connexin-interacting proteins

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  8. The dynamic multisite interactions between two intrinsically disordered proteins

    Wu, Shaowen; Wang, Dongdong; Liu, Jin; Feng, Yitao; Weng, Jingwei; Li, Yu; Gao, Xin; Liu, Jianwei; Wang, Wenning

    2017-01-01

    Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well characterized folding upon binding to dynamic fuzzy complex. To date, most studies concern the binding of an IDP to a

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

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

    2017-08-01

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

  10. Identification of proteins that may directly interact with human RPA.

    Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

    2010-11-01

    RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

  11. A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase

    Dimitrios Vlachakis

    2013-05-01

    Full Text Available Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12 was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost “U” shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.

  12. quinolinium iodide in suppression of protein–protein interactions

    In searching for alternative ways to reduce protein–protein interactions or to inhibit the amyloid formation, the inhibitory effects ..... ing the exposure of hydrophobic surfaces mirrors the ... is well-supported by electrostatic interactions between.

  13. HKC: An Algorithm to Predict Protein Complexes in Protein-Protein Interaction Networks

    Xiaomin Wang

    2011-01-01

    Full Text Available 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.

  14. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

    2013-09-03

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

  15. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

    2013-01-01

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

  16. Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

    Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

    2018-01-15

    The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

  17. Proteins interacting with the 26S proteasome

    Hartmann-Petersen, R; Gordon, C

    2004-01-01

    The 26S proteasome is the multi-protein protease that recognizes and degrades ubiquitinylated substrates targeted for destruction by the ubiquitin pathway. In addition to the well-documented subunit organization of the 26S holoenzyme, it is clear that a number of other proteins transiently...... associate with the 26S complex. These transiently associated proteins confer a number of different roles such as substrate presentation, cleavage of the multi-ubiquitin chain from the protein substrate and turnover of misfolded proteins. Such activities are essential for the 26S proteasome to efficiently...... fulfill its intracellular function in protein degradation....

  18. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  19. PPI-IRO: A two-stage method for protein-protein interaction extraction based on interaction relation ontology

    Li, Chuanxi; Chen, Peng; Wang, Rujing; Wang, Xiujie; Su, Yaru; Li, Jinyan

    2014-01-01

    Mining Protein-Protein Interactions (PPIs) from the fast-growing biomedical literature resources has been proven as an effective approach for the identifi cation of biological regulatory networks. This paper presents a novel method based on the idea

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

    Kaare eTeilum

    2015-07-01

    Full Text Available 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.

  1. The dynamic multisite interactions between two intrinsically disordered proteins

    Wu, Shaowen

    2017-05-11

    Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well characterized folding upon binding to dynamic fuzzy complex. To date, most studies concern the binding of an IDP to a structured protein, while the Interaction between two IDPs is poorly understood. In this study, we combined NMR, smFRET, and molecular dynamics (MD) simulation to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by MD and mutagenesis studies. Our study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.

  2. Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins ▿#

    Cady, Kyle C.; O'Toole, George A.

    2011-01-01

    Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria. PMID:21398535

  3. Core Data of Yeast Interacting Proteins Database (Original Version) - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Full Text Available y are in the reverse direction. *1 A comprehensive two-hybrid analysis to explore the yeast protein interact...s. 2000 Jan 1;28(1):73-6. *2 The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive...000 Jan 1;28(1):73-6. *3 A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisia

  4. BRCA1 interacts directly with the Fanconi anemia protein FANCA.

    Folias, Alexandra; Matkovic, Mara; Bruun, Donald; Reid, Sonja; Hejna, James; Grompe, Markus; D'Andrea, Alan; Moses, Robb

    2002-10-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.

  5. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  6. Protein interactions in genome maintenance as novel antibacterial targets.

    Aimee H Marceau

    Full Text Available 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.

  7. The simulation approach to lipid-protein interactions.

    Paramo, Teresa; Garzón, Diana; Holdbrook, Daniel A; Khalid, Syma; Bond, Peter J

    2013-01-01

    The interactions between lipids and proteins are crucial for a range of biological processes, from the folding and stability of membrane proteins to signaling and metabolism facilitated by lipid-binding proteins. However, high-resolution structural details concerning functional lipid/protein interactions are scarce due to barriers in both experimental isolation of native lipid-bound complexes and subsequent biophysical characterization. The molecular dynamics (MD) simulation approach provides a means to complement available structural data, yielding dynamic, structural, and thermodynamic data for a protein embedded within a physiologically realistic, modelled lipid environment. In this chapter, we provide a guide to current methods for setting up and running simulations of membrane proteins and soluble, lipid-binding proteins, using standard atomistically detailed representations, as well as simplified, coarse-grained models. In addition, we outline recent studies that illustrate the power of the simulation approach in the context of biologically relevant lipid/protein interactions.

  8. Prediction of protein–protein interactions: unifying evolution and structure at protein interfaces

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-01-01

    The vast majority of the chores in the living cell involve protein–protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein–protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations

  9. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    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

    2012-07-01

    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.

  10. [RTEL1 (regulator of telomere elongation helicase 1), a DNA helicase essential for genome stability].

    Le Guen, Tangui; Jullien, Laurent; Schertzer, Mike; Lefebvre, Axelle; Kermasson, Laetitia; de Villartay, Jean-Pierre; Londoño-Vallejo, Arturo; Revy, Patrick

    2013-12-01

    RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play. © 2013 médecine/sciences – Inserm.

  11. Hot-spot analysis for drug discovery targeting protein-protein interactions.

    Rosell, Mireia; Fernández-Recio, Juan

    2018-04-01

    Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

  12. A Physical Interaction Network of Dengue Virus and Human Proteins*

    Khadka, Sudip; Vangeloff, Abbey D.; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S.; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J.; Perera, Rushika; LaCount, Douglas J.

    2011-01-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection. PMID:21911577

  13. A physical interaction network of dengue virus and human proteins.

    Khadka, Sudip; Vangeloff, Abbey D; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J; Perera, Rushika; LaCount, Douglas J

    2011-12-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

  14. Interactions among tobacco sieve element occlusion (SEO) proteins.

    Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

    2012-12-01

    Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

  15. hSSB1 associates with and promotes stability of the BLM helicase

    O'BYRNE, KEN

    2017-01-01

    Background Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. Results In this s...

  16. Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

    Wang, Weiping; Manna, David; Simmons, Daniel T

    2007-05-01

    The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

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

    Teilum, Kaare; Olsen, Johan Gotthardt; Kragelund, Birthe Brandt

    2015-01-01

    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)....

  18. Mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays

    Rigter, A.; Priem, J.; Timmers-Parohi, D.; Langeveld, J.; Bossers, A.

    2009-01-01

    Protein-protein interactions are at the basis of most if not all biological processes in living cells. Therefore, adapting existing techniques or developing new techniques to study interactions between proteins are of importance in elucidating which amino acid sequences contribute to these

  19. Imaging protein-protein interactions in living cells

    Hink, M.A.; Bisseling, T.; Visser, A.J.W.G.

    2002-01-01

    The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide

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

    Park, Byungkyu; Han, Kyungsook

    2010-01-18

    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. 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. 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.

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

    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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Structural study of surfactant-dependent interaction with protein

    Mehan, Sumit; Aswal, Vinod K., E-mail: vkaswal@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, Joachim [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)

    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.

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

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Protein-lipid interactions: from membrane domains to cellular networks

    Tamm, Lukas K

    2005-01-01

    ... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...

  5. Interaction between Protein, Phytate, and Microbial Phytase. In Vitro Studies

    Kies, A.K.; Jonge, de L.H.; Kemme, P.A.; Jongbloed, A.W.

    2006-01-01

    The interaction between protein and phytate was investigated in vitro using proteins extracted from five common feedstuffs and from casein. The appearance of naturally present soluble protein-phytate complexes in the feedstuffs, the formation of complexes at different pHs, and the degradation of

  6. The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities

    Mandy Jeske

    2015-07-01

    Full Text Available In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

  7. Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

    Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I

    2014-01-01

    Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathologic...

  8. Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

    Boskovic, Jasminka; Bragado-Nilsson, Elisabeth; Saligram Prabhakar, Bhargav

    2016-01-01

    DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replicati...

  9. NatalieQ: A web server for protein-protein interaction network querying

    El-Kebir, M.; Brandt, B.W.; Heringa, J.; Klau, G.W.

    2014-01-01

    Background Molecular interactions need to be taken into account to adequately model the complex behavior of biological systems. These interactions are captured by various types of biological networks, such as metabolic, gene-regulatory, signal transduction and protein-protein interaction networks.

  10. Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

    Kunkel, Thomas A; Burgers, Peter M J

    2017-08-01

    Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  11. Casein - whey protein interactions in heated milk

    Vasbinder, Astrid Jolanda

    2002-01-01

    Heating of milk is an essential step in the processing of various dairy products, like for example yoghurt. A major consequence of the heat treatment is the denaturation of whey proteins, which either associate with the casein micelle or form soluble whey protein aggregates. By combination of

  12. The role of electrostatics in protein-protein interactions of a monoclonal antibody.

    Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

    2014-07-07

    Understanding how protein-protein interactions depend on the choice of buffer, salt, ionic strength, and pH is needed to have better control over protein solution behavior. Here, we have characterized the pH and ionic strength dependence of protein-protein interactions in terms of an interaction parameter kD obtained from dynamic light scattering and the osmotic second virial coefficient B22 measured by static light scattering. A simplified protein-protein interaction model based on a Baxter adhesive potential and an electric double layer force is used to separate out the contributions of longer-ranged electrostatic interactions from short-ranged attractive forces. The ionic strength dependence of protein-protein interactions for solutions at pH 6.5 and below can be accurately captured using a Deryaguin-Landau-Verwey-Overbeek (DLVO) potential to describe the double layer forces. In solutions at pH 9, attractive electrostatics occur over the ionic strength range of 5-275 mM. At intermediate pH values (7.25 to 8.5), there is a crossover effect characterized by a nonmonotonic ionic strength dependence of protein-protein interactions, which can be rationalized by the competing effects of long-ranged repulsive double layer forces at low ionic strength and a shorter ranged electrostatic attraction, which dominates above a critical ionic strength. The change of interactions from repulsive to attractive indicates a concomitant change in the angular dependence of protein-protein interaction from isotropic to anisotropic. In the second part of the paper, we show how the Baxter adhesive potential can be used to predict values of kD from fitting to B22 measurements, thus providing a molecular basis for the linear correlation between the two protein-protein interaction parameters.

  13. Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

    Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

    2013-01-01

    We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

  14. Dendrimer-protein interactions versus dendrimer-based nanomedicine.

    Shcharbin, Dzmitry; Shcharbina, Natallia; Dzmitruk, Volha; Pedziwiatr-Werbicka, Elzbieta; Ionov, Maksim; Mignani, Serge; de la Mata, F Javier; Gómez, Rafael; Muñoz-Fernández, Maria Angeles; Majoral, Jean-Pierre; Bryszewska, Maria

    2017-04-01

    Dendrimers are hyperbranched polymers belonging to the huge class of nanomedical devices. Their wide application in biology and medicine requires understanding of the fundamental mechanisms of their interactions with biological systems. Summarizing, electrostatic force plays the predominant role in dendrimer-protein interactions, especially with charged dendrimers. Other kinds of interactions have been proven, such as H-bonding, van der Waals forces, and even hydrophobic interactions. These interactions depend on the characteristics of both participants: flexibility and surface charge of a dendrimer, rigidity of protein structure and the localization of charged amino acids at its surface. pH and ionic strength of solutions can significantly modulate interactions. Ligands and cofactors attached to a protein can also change dendrimer-protein interactions. Binding of dendrimers to a protein can change its secondary structure, conformation, intramolecular mobility and functional activity. However, this strongly depends on rigidity versus flexibility of a protein's structure. In addition, the potential applications of dendrimers to nanomedicine are reviwed related to dendrimer-protein interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing

    Easton, Douglas Frederick; Lesueur, Fabienne; Decker, Brennan; Michailidou, Kyriaki; Li, Jun; Allen, Jamie; Luccarini, Craig; Pooley, Karen Anne; Shah, Mitulkumar Nandlal; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Ahmad, Jamil; Thompson, Ella R; Damiola, Francesca

    2016-01-01

    Background BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. Metho...

  16. Interaction between -Synuclein and Other Proteins in Neurodegenerative Disorders

    Kurt A. Jellinger

    2011-01-01

    Full Text Available Protein aggregation is a common characteristic of many neurodegenerative disorders, and the interaction between pathological/toxic proteins to cause neurodegeneration is a hot topic of current neuroscience research. Despite clinical, genetic, and experimental differences, evidence increasingly indicates considerable overlap between synucleinopathies and tauopathies or other protein-misfolding diseases. Inclusions, characteristics of these disorders, also occurring in other neurodegenerative diseases, suggest interactions of pathological proteins engaging common downstream pathways. Novel findings that have shifted our understanding in the role of pathologic proteins in the pathogenesis of Parkinson and Alzheimer diseases have confirmed correlations/overlaps between these and other neurodegenerative disorders. The synergistic effects of α-synuclein, hyperphosphorylated tau, amyloid-β, and other pathologic proteins, and the underlying molecular pathogenic mechanisms, including induction and spread of protein aggregates, are critically reviewed, suggesting a dualism or triad of neurodegeneration in protein-misfolding disorders, although the etiology of most of these processes is still mysterious.

  17. Protein-material interactions: From micro-to-nano scale

    Tsapikouni, Theodora S.; Missirlis, Yannis F.

    2008-01-01

    The article presents a survey on the significance of protein-material interactions, the mechanisms which control them and the techniques used for their study. Protein-surface interactions play a key role in regenerative medicine, drug delivery, biosensor technology and chromatography, while it is related to various undesired effects such as biofouling and bio-prosthetic malfunction. Although the effects of protein-surface interaction concern the micro-scale, being sometimes obvious even with bare eyes, they derive from biophysical events at the nano-scale. The sequential steps for protein adsorption involve events at the single biomolecule level and the forces driving or inhibiting protein adsorption act at the molecular level too. Following the scaling of protein-surface interactions, various techniques have been developed for their study both in the micro- and nano-scale. Protein labelling with radioisotopes or fluorescent probes, colorimetric assays and the quartz crystal microbalance were the first techniques used to monitor protein adsorption isotherms, while the surface force apparatus was used to measure the interaction forces between protein layers at the micro-scale. Recently, more elaborate techniques like total internal reflection fluorescence (TIRF), Fourier transform infrared spectroscopy (FTIR), surface plasmon resonance, Raman spectroscopy, ellipsometry and time of flight secondary ion mass spectrometry (ToF-SIMS) have been applied for the investigation of protein density, structure or orientation at the interfaces. However, a turning point in the study of protein interactions with the surfaces was the invention and the wide-spread use of atomic force microscopy (AFM) which can both image single protein molecules on surfaces and directly measure the interaction force

  18. Structural basis of Zika virus helicase in recognizing its substrates

    Hongliang Tian

    2016-07-01

    Full Text Available Abstract The recent explosive outbreak of Zika virus (ZIKV infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

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

    Carson M Andorf

    Full Text Available Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners.Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  20. A credit-card library approach for disrupting protein-protein interactions.

    Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

    2006-04-15

    Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

  1. Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions.

    Meckes, David G

    2014-01-01

    The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

  2. Unveiling protein functions through the dynamics of the interaction network.

    Irene Sendiña-Nadal

    Full Text Available Protein interaction networks have become a tool to study biological processes, either for predicting molecular functions or for designing proper new drugs to regulate the main biological interactions. Furthermore, such networks are known to be organized in sub-networks of proteins contributing to the same cellular function. However, the protein function prediction is not accurate and each protein has traditionally been assigned to only one function by the network formalism. By considering the network of the physical interactions between proteins of the yeast together with a manual and single functional classification scheme, we introduce a method able to reveal important information on protein function, at both micro- and macro-scale. In particular, the inspection of the properties of oscillatory dynamics on top of the protein interaction network leads to the identification of misclassification problems in protein function assignments, as well as to unveil correct identification of protein functions. We also demonstrate that our approach can give a network representation of the meta-organization of biological processes by unraveling the interactions between different functional classes.

  3. Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

    Emilie eGauliard

    2015-02-01

    Full Text Available Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins. Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

  4. Alignment of non-covalent interactions at protein-protein interfaces.

    Hongbo Zhu

    Full Text Available BACKGROUND: The study and comparison of protein-protein interfaces is essential for the understanding of the mechanisms of interaction between proteins. While there are many methods for comparing protein structures and protein binding sites, so far no methods have been reported for comparing the geometry of non-covalent interactions occurring at protein-protein interfaces. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a method for aligning non-covalent interactions between different protein-protein interfaces. The method aligns the vector representations of van der Waals interactions and hydrogen bonds based on their geometry. The method has been applied to a dataset which comprises a variety of protein-protein interfaces. The alignments are consistent to a large extent with the results obtained using two other complementary approaches. In addition, we apply the method to three examples of protein mimicry. The method successfully aligns respective interfaces and allows for recognizing conserved interface regions. CONCLUSIONS/SIGNIFICANCE: The Galinter method has been validated in the comparison of interfaces in which homologous subunits are involved, including cases of mimicry. The method is also applicable to comparing interfaces involving non-peptidic compounds. Galinter assists users in identifying local interface regions with similar patterns of non-covalent interactions. This is particularly relevant to the investigation of the molecular basis of interaction mimicry.

  5. A protein domain interaction interface database: InterPare

    Lee Jungsul

    2005-08-01

    Full Text Available Abstract Background Most proteins function by interacting with other molecules. Their interaction interfaces are highly conserved throughout evolution to avoid undesirable interactions that lead to fatal disorders in cells. Rational drug discovery includes computational methods to identify the interaction sites of lead compounds to the target molecules. Identifying and classifying protein interaction interfaces on a large scale can help researchers discover drug targets more efficiently. Description We introduce a large-scale protein domain interaction interface database called InterPare http://interpare.net. It contains both inter-chain (between chains interfaces and intra-chain (within chain interfaces. InterPare uses three methods to detect interfaces: 1 the geometric distance method for checking the distance between atoms that belong to different domains, 2 Accessible Surface Area (ASA, a method for detecting the buried region of a protein that is detached from a solvent when forming multimers or complexes, and 3 the Voronoi diagram, a computational geometry method that uses a mathematical definition of interface regions. InterPare includes visualization tools to display protein interior, surface, and interaction interfaces. It also provides statistics such as the amino acid propensities of queried protein according to its interior, surface, and interface region. The atom coordinates that belong to interface, surface, and interior regions can be downloaded from the website. Conclusion InterPare is an open and public database server for protein interaction interface information. It contains the large-scale interface data for proteins whose 3D-structures are known. As of November 2004, there were 10,583 (Geometric distance, 10,431 (ASA, and 11,010 (Voronoi diagram entries in the Protein Data Bank (PDB containing interfaces, according to the above three methods. In the case of the geometric distance method, there are 31,620 inter-chain domain

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

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

    2016-02-15

    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. 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 protein interaction

  7. From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

    Mu Gao

    2009-03-01

    Full Text Available 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 Calpha deviation from native is up to 5 A 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.

  8. Prediction of protein-protein interactions between viruses and human by an SVM model

    Cui Guangyu

    2012-05-01

    Full Text Available Abstract Background Several computational methods have been developed to predict protein-protein interactions from amino acid sequences, but most of those methods are intended for the interactions within a species rather than for interactions across different species. Methods for predicting interactions between homogeneous proteins are not appropriate for finding those between heterogeneous proteins since they do not distinguish the interactions between proteins of the same species from those of different species. Results We developed a new method for representing a protein sequence of variable length in a frequency vector of fixed length, which encodes the relative frequency of three consecutive amino acids of a sequence. We built a support vector machine (SVM model to predict human proteins that interact with virus proteins. In two types of viruses, human papillomaviruses (HPV and hepatitis C virus (HCV, our SVM model achieved an average accuracy above 80%, which is higher than that of another SVM model with a different representation scheme. Using the SVM model and Gene Ontology (GO annotations of proteins, we predicted new interactions between virus proteins and human proteins. Conclusions Encoding the relative frequency of amino acid triplets of a protein sequence is a simple yet powerful representation method for predicting protein-protein interactions across different species. The representation method has several advantages: (1 it enables a prediction model to achieve a better performance than other representations, (2 it generates feature vectors of fixed length regardless of the sequence length, and (3 the same representation is applicable to different types of proteins.

  9. Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

    Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

    2017-06-02

    Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

  10. Novel Technology for Protein-Protein Interaction-based Targeted Drug Discovery

    Jung Me Hwang

    2011-12-01

    Full Text Available We have developed a simple but highly efficient in-cell protein-protein interaction (PPI discovery system based on the translocation properties of protein kinase C- and its C1a domain in live cells. This system allows the visual detection of trimeric and dimeric protein interactions including cytosolic, nuclear, and/or membrane proteins with their cognate ligands. In addition, this system can be used to identify pharmacological small compounds that inhibit specific PPIs. These properties make this PPI system an attractive tool for screening drug candidates and mapping the protein interactome.

  11. Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

    Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka; Pigli, Ying Z.; Boyle-Vavra, Susan; Rice , Phoebe A. (UC)

    2016-08-29

    Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

  12. Protein prenylation: a new mode of host-pathogen interaction.

    Amaya, Moushimi; Baranova, Ancha; van Hoek, Monique L

    2011-12-09

    Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Anomalous Protein-Protein Interactions in Multivalent Salt Solution

    Pasquier, C.; Vazdar, M.; Forsman, J.; Jungwirth, Pavel; Lund, M.

    2017-01-01

    Roč. 121, č. 14 (2017), s. 3000-3006 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-01074S Institutional support: RVO:61388963 Keywords : Monte Carlo * molecular dynamics * membranes * proteins * multivalent salts Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 3.177, year: 2016

  14. A protein interaction map of the kalimantacin biosynthesis assembly line

    Birgit Uytterhoeven

    2016-11-01

    Full Text Available The antimicrobial secondary metabolite kalimantacin is produced by a hybrid polyketide/ non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites.This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.

  15. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

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

    2011-01-01

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

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

    Chowdhury, Saiful M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shi, Liang [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Yoon, Hyunjin [Dartmouth College, Hanover, NH (United States); Ansong, Charles [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rommereim, Leah M. [Dartmouth College, Hanover, NH (United States); Norbeck, Angela D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Auberry, Kenneth J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Moore, R. J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Adkins, Joshua N. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Heffron, Fred [Oregon Health and Science Univ., Portland, OR (United States); Smith, Richard D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    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. .

  17. Quality control methodology for high-throughput protein-protein interaction screening.

    Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

    2011-01-01

    Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

  18. Reciprocal carbonyl-carbonyl interactions in small molecules and proteins.

    Rahim, Abdur; Saha, Pinaki; Jha, Kunal Kumar; Sukumar, Nagamani; Sarma, Bani Kanta

    2017-07-19

    Carbonyl-carbonyl n→π* interactions where a lone pair (n) of the oxygen atom of a carbonyl group is delocalized over the π* orbital of a nearby carbonyl group have attracted a lot of attention in recent years due to their ability to affect the 3D structure of small molecules, polyesters, peptides, and proteins. In this paper, we report the discovery of a "reciprocal" carbonyl-carbonyl interaction with substantial back and forth n→π* and π→π* electron delocalization between neighboring carbonyl groups. We have carried out experimental studies, analyses of crystallographic databases and theoretical calculations to show the presence of this interaction in both small molecules and proteins. In proteins, these interactions are primarily found in polyproline II (PPII) helices. As PPII are the most abundant secondary structures in unfolded proteins, we propose that these local interactions may have implications in protein folding.Carbonyl-carbonyl π* non covalent interactions affect the structure and stability of small molecules and proteins. Here, the authors carry out experimental studies, analyses of crystallographic databases and theoretical calculations to describe an additional type of carbonyl-carbonyl interaction.

  19. Specificity and evolvability in eukaryotic protein interaction networks.

    Pedro Beltrao

    2007-02-01

    Full Text Available Progress in uncovering the protein interaction networks of several species has led to questions of what underlying principles might govern their organization. Few studies have tried to determine the impact of protein interaction network evolution on the observed physiological differences between species. Using comparative genomics and structural information, we show here that eukaryotic species have rewired their interactomes at a fast rate of approximately 10(-5 interactions changed per protein pair, per million years of divergence. For Homo sapiens this corresponds to 10(3 interactions changed per million years. Additionally we find that the specificity of binding strongly determines the interaction turnover and that different biological processes show significantly different link dynamics. In particular, human proteins involved in immune response, transport, and establishment of localization show signs of positive selection for change of interactions. Our analysis suggests that a small degree of molecular divergence can give rise to important changes at the network level. We propose that the power law distribution observed in protein interaction networks could be partly explained by the cell's requirement for different degrees of protein binding specificity.

  20. Evolutionary diversification of protein-protein interactions by interface add-ons.

    Plach, Maximilian G; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H; Merkl, Rainer; Sterner, Reinhard

    2017-10-03

    Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

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

    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

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

    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

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

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

    2011-01-01

    Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → 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, β-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.

  4. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes.

    Jiawei Luo

    Full Text Available Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC, based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID, of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.Experimental results based on three different PPI(protein-protein interaction networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC.LIDC is more effective for the prediction of essential proteins than other recently developed methods.

  5. (S)Pinning down protein interactions by NMR

    Teilum, Kaare; Kunze, Micha Ben Achim; Erlendsson, Simon

    2017-01-01

    Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions...... all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative...... descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background...

  6. Membrane interaction of retroviral Gag proteins

    Robert Alfred Dick

    2014-04-01

    Full Text Available 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.

  7. Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction.

    Aalt D J van Dijk

    Full Text Available 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

  8. A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

    Ida Nielsen

    Full Text Available The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3. Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

  9. Predicting protein-protein interactions from multimodal biological data sources via nonnegative matrix tri-factorization.

    Wang, Hua; Huang, Heng; Ding, Chris; Nie, Feiping

    2013-04-01

    Protein interactions are central to all the biological processes and structural scaffolds in living organisms, because they orchestrate a number of cellular processes such as metabolic pathways and immunological recognition. Several high-throughput methods, for example, yeast two-hybrid system and mass spectrometry method, can help determine protein interactions, which, however, suffer from high false-positive rates. Moreover, many protein interactions predicted by one method are not supported by another. Therefore, computational methods are necessary and crucial to complete the interactome expeditiously. In this work, we formulate the problem of predicting protein interactions from a new mathematical perspective--sparse matrix completion, and propose a novel nonnegative matrix factorization (NMF)-based matrix completion approach to predict new protein interactions from existing protein interaction networks. Through using manifold regularization, we further develop our method to integrate different biological data sources, such as protein sequences, gene expressions, protein structure information, etc. Extensive experimental results on four species, Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, and Caenorhabditis elegans, have shown that our new methods outperform related state-of-the-art protein interaction prediction methods.

  10. Protein-protein interactions in the regulation of WRKY transcription factors.

    Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-03-01

    It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

  11. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

    Blagoev, B.; Kratchmarova, I.; Ong, S.E.

    2003-01-01

    Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we em...

  12. Filtering high-throughput protein-protein interaction data using a combination of genomic features

    Patil Ashwini

    2005-04-01

    Full Text Available Abstract Background Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. Results In this study, we use a combination of 3 genomic features – structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology – as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90% and good specificity (63%. We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. Conclusion A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

  13. Protein complex prediction based on k-connected subgraphs in protein interaction network

    Habibi Mahnaz

    2010-09-01

    Full Text Available Abstract Background Protein complexes play an important role in cellular mechanisms. Recently, several methods have been presented to predict protein complexes in a protein interaction network. In these methods, a protein complex is predicted as a dense subgraph of protein interactions. However, interactions data are incomplete and a protein complex does not have to be a complete or dense subgraph. Results We propose a more appropriate protein complex prediction method, CFA, that is based on connectivity number on subgraphs. We evaluate CFA using several protein interaction networks on reference protein complexes in two benchmark data sets (MIPS and Aloy, containing 1142 and 61 known complexes respectively. We compare CFA to some existing protein complex prediction methods (CMC, MCL, PCP and RNSC in terms of recall and precision. We show that CFA predicts more complexes correctly at a competitive level of precision. Conclusions Many real complexes with different connectivity level in protein interaction network can be predicted based on connectivity number. Our CFA program and results are freely available from http://www.bioinf.cs.ipm.ir/softwares/cfa/CFA.rar.

  14. Yeast Interacting Proteins Database: YNR006W, YHL002W [Yeast Interacting Proteins Database

    Full Text Available ling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation; ..., as well as for recycling of Golgi proteins and formation of lumenal membranes Rows with this prey as prey ...1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins dest...degradation, as well as for recycling of Golgi proteins and formation of lumenal membranes

  15. Improving accuracy of protein-protein interaction prediction by considering the converse problem for sequence representation

    Wang Yong

    2011-10-01

    Full Text Available Abstract Background With the development of genome-sequencing technologies, protein sequences are readily obtained by translating the measured mRNAs. Therefore predicting protein-protein interactions from the sequences is of great demand. The reason lies in the fact that identifying protein-protein interactions is becoming a bottleneck for eventually understanding the functions of proteins, especially for those organisms barely characterized. Although a few methods have been proposed, the converse problem, if the features used extract sufficient and unbiased information from protein sequences, is almost untouched. Results In this study, we interrogate this problem theoretically by an optimization scheme. Motivated by the theoretical investigation, we find novel encoding methods for both protein sequences and protein pairs. Our new methods exploit sufficiently the information of protein sequences and reduce artificial bias and computational cost. Thus, it significantly outperforms the available methods regarding sensitivity, specificity, precision, and recall with cross-validation evaluation and reaches ~80% and ~90% accuracy in Escherichia coli and Saccharomyces cerevisiae respectively. Our findings here hold important implication for other sequence-based prediction tasks because representation of biological sequence is always the first step in computational biology. Conclusions By considering the converse problem, we propose new representation methods for both protein sequences and protein pairs. The results show that our method significantly improves the accuracy of protein-protein interaction predictions.

  16. Quantification of protein interaction kinetics in a micro droplet

    Yin, L. L. [Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, Arizona 85287 (United States); College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Wang, S. P., E-mail: shaopeng.wang@asu.edu, E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J., E-mail: shaopeng.wang@asu.edu, E-mail: njtao@asu.edu [Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, Arizona 85287 (United States); Zhang, S. T. [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China)

    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.

  17. ProteinShop: A tool for interactive protein manipulation and steering

    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.

  18. Understanding Protein Synthesis: An Interactive Card Game Discussion

    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…

  19. Interaction of maize chromatin-associated HMG proteins with mononucleosomes

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...

  20. Prediction of localization and interactions of apoptotic proteins

    Matula Pavel

    2009-07-01

    Full Text Available Abstract During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF and endonuclease G (endoG. Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID. We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

  1. PIPE: a protein-protein interaction prediction engine based on the re-occurring short polypeptide sequences between known interacting protein pairs

    Greenblatt Jack

    2006-07-01

    Full Text Available Abstract Background Identification of protein interaction networks has received considerable attention in the post-genomic era. The currently available biochemical approaches used to detect protein-protein interactions are all time and labour intensive. Consequently there is a growing need for the development of computational tools that are capable of effectively identifying such interactions. Results Here we explain the development and implementation of a novel Protein-Protein Interaction Prediction Engine termed PIPE. This tool is capable of predicting protein-protein interactions for any target pair of the yeast Saccharomyces cerevisiae proteins from their primary structure and without the need for any additional information or predictions about the proteins. PIPE showed a sensitivity of 61% for detecting any yeast protein interaction with 89% specificity and an overall accuracy of 75%. This rate of success is comparable to those associated with the most commonly used biochemical techniques. Using PIPE, we identified a novel interaction between YGL227W (vid30 and YMR135C (gid8 yeast proteins. This lead us to the identification of a novel yeast complex that here we term vid30 complex (vid30c. The observed interaction was confirmed by tandem affinity purification (TAP tag, verifying the ability of PIPE to predict novel protein-protein interactions. We then used PIPE analysis to investigate the internal architecture of vid30c. It appeared from PIPE analysis that vid30c may consist of a core and a secondary component. Generation of yeast gene deletion strains combined with TAP tagging analysis indicated that the deletion of a member of the core component interfered with the formation of vid30c, however, deletion of a member of the secondary component had little effect (if any on the formation of vid30c. Also, PIPE can be used to analyse yeast proteins for which TAP tagging fails, thereby allowing us to predict protein interactions that are not

  2. Catching the PEG-induced attractive interaction between proteins.

    Vivarès, D; Belloni, L; Tardieu, A; Bonneté, F

    2002-09-01

    We present the experimental and theoretical background of a method to characterize the protein-protein attractive potential induced by one of the mostly used crystallizing agents in the protein-field, the poly(ethylene glycol) (PEG). This attractive interaction is commonly called, in colloid physics, the depletion interaction. Small-Angle X-ray Scattering experiments and numerical treatments based on liquid-state theories were performed on urate oxidase-PEG mixtures with two different PEGs (3350 Da and 8000 Da). A "two-component" approach was used in which the polymer-polymer, the protein-polymer and the protein-protein pair potentials were determined. The resulting effective protein-protein potential was characterized. This potential is the sum of the free-polymer protein-protein potential and of the PEG-induced depletion potential. The depletion potential was found to be hardly dependent upon the protein concentration but strongly function of the polymer size and concentration. Our results were also compared with two models, which give an analytic expression for the depletion potential.

  3. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

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

    Benjamin J. Forred

    2016-01-01

    Full Text Available 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.

  5. NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

    Zhou, Ting; Ren, Xiaoming; Adams, Rebecca L.; Pyle, Anna Marie; Ou, J. -H. James

    2017-10-25

    Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.

    IMPORTANCEGenotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important

  6. Prediction of heterodimeric protein complexes from weighted protein-protein interaction networks using novel features and kernel functions.

    Peiying Ruan

    Full Text Available Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes.

  7. Structural interface parameters are discriminatory in recognising near-native poses of protein-protein interactions.

    Sony Malhotra

    Full Text Available Interactions at the molecular level in the cellular environment play a very crucial role in maintaining the physiological functioning of the cell. These molecular interactions exist at varied levels viz. protein-protein interactions, protein-nucleic acid interactions or protein-small molecules interactions. Presently in the field, these interactions and their mechanisms mark intensively studied areas. Molecular interactions can also be studied computationally using the approach named as Molecular Docking. Molecular docking employs search algorithms to predict the possible conformations for interacting partners and then calculates interaction energies. However, docking proposes number of solutions as different docked poses and hence offers a serious challenge to identify the native (or near native structures from the pool of these docked poses. Here, we propose a rigorous scoring scheme called DockScore which can be used to rank the docked poses and identify the best docked pose out of many as proposed by docking algorithm employed. The scoring identifies the optimal interactions between the two protein partners utilising various features of the putative interface like area, short contacts, conservation, spatial clustering and the presence of positively charged and hydrophobic residues. DockScore was first trained on a set of 30 protein-protein complexes to determine the weights for different parameters. Subsequently, we tested the scoring scheme on 30 different protein-protein complexes and native or near-native structure were assigned the top rank from a pool of docked poses in 26 of the tested cases. We tested the ability of DockScore to discriminate likely dimer interactions that differ substantially within a homologous family and also demonstrate that DOCKSCORE can distinguish correct pose for all 10 recent CAPRI targets.

  8. Evolution of a protein domain interaction network

    Li-Feng, Gao; Jian-Jun, Shi; Shan, Guan

    2010-01-01

    In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows: (i) there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; (ii) few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; (iii) in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases. (general)

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

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

    2004-03-15

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

  10. Exploiting conformational ensembles in modeling protein-protein interactions on the proteome scale

    Kuzu, Guray; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

    2013-01-01

    Cellular functions are performed through protein-protein interactions; therefore, identification of these interactions is crucial for understanding biological processes. Recent studies suggest that knowledge-based approaches are more useful than ‘blind’ docking for modeling at large scales. However, a caveat of knowledge-based approaches is that they treat molecules as rigid structures. The Protein Data Bank (PDB) offers a wealth of conformations. Here, we exploited ensemble of the conformations in predictions by a knowledge-based method, PRISM. We tested ‘difficult’ cases in a docking-benchmark dataset, where the unbound and bound protein forms are structurally different. Considering alternative conformations for each protein, the percentage of successfully predicted interactions increased from ~26% to 66%, and 57% of the interactions were successfully predicted in an ‘unbiased’ scenario, in which data related to the bound forms were not utilized. If the appropriate conformation, or relevant template interface, is unavailable in the PDB, PRISM could not predict the interaction successfully. The pace of the growth of the PDB promises a rapid increase of ensemble conformations emphasizing the merit of such knowledge-based ensemble strategies for higher success rates in protein-protein interaction predictions on an interactome-scale. We constructed the structural network of ERK interacting proteins as a case study. PMID:23590674

  11. Understanding protein–protein interactions by genetic suppression

    Unknown

    Protein–protein interactions influence many cellular processes and it is increasingly being felt that even a weak and ... In a bacterial system where the complete genome sequence is available, it is an arduous ... teins (primary mutations) are useful in these studies. ... of interaction of this antibiotic with the central enzyme.

  12. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

    Mehrnoush Dehghani

    2015-03-01

    Full Text Available The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells, posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

  13. Identification of structural protein-protein interactions of herpes simplex virus type 1.

    Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

    2008-09-01

    In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

  14. [Detection of protein-protein interactions by FRET and BRET methods].

    Matoulková, E; Vojtěšek, B

    2014-01-01

    Nowadays, in vivo protein-protein interaction studies have become preferable detecting meth-ods that enable to show or specify (already known) protein interactions and discover their inhibitors. They also facilitate detection of protein conformational changes and discovery or specification of signaling pathways in living cells. One group of in vivo methods enabling these findings is based on fluorescent resonance energy transfer (FRET) and its bio-luminescent modification (BRET). They are based on visualization of protein-protein interactions via light or enzymatic excitation of fluorescent or bio-luminescent proteins. These methods allow not only protein localization within the cell or its organelles (or small animals) but they also allow us to quantify fluorescent signals and to discover weak or strong interaction partners. In this review, we explain the principles of FRET and BRET, their applications in the characterization of protein-protein interactions and we describe several findings using these two methods that clarify molecular and cellular mechanisms and signals related to cancer biology.

  15. Protein-protein interaction inference based on semantic similarity of Gene Ontology terms.

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Structural basis for the function of DEAH helicases

    He, Yangzi; Andersen, Gregers Rom; Nielsen, Klaus Hvid

    2010-01-01

    DEAH helicases participate in pre‐messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p‐ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide‐binding motif. A β‐hairpin from the second RecA domain is wedged between two carboxy......‐terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C‐terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides...

  17. Foot-printing of Protein Interactions by Tritium Labeling

    Mousseau, Guillaume; Thomas, Olivier P.; Agez, Morgane; Thai, Robert; Cintrat, Jean-Christophe; Rousseau, Bernard; Raffy, Quentin; Renault, Jean Philippe; Pin, Serge; Ochsenbein, Francoise

    2010-01-01

    A new foot-printing method for mapping protein interactions has been developed, using tritium as a radioactive label. As residues involved in an interaction are less labeled when the complex is formed, they can be identified via comparison of the tritium incorporation of each residue of the bound protein with that of the unbound one. Application of this foot-printing method to the complex formed by the histone H3 fragment H3 122-135 and the protein hAsflA 1-156 afforded data in good agreement with NMR results. (authors)

  18. Quantitative analysis of protein-ligand interactions by NMR.

    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

  19. A selection that reports on protein-protein interactions within a thermophilic bacterium.

    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.

  20. Yeast Interacting Proteins Database: YHL002W, YNR006W [Yeast Interacting Proteins Database

    Full Text Available ycling of Golgi proteins and formation of lumenal membranes Rows with this bait as bait (1) Rows with this b...required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined...on, as well as for recycling of Golgi proteins and formation of lumenal membranes...ith Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated protei

  1. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

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

    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.

  3. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    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.

  4. Direct measurements of protein-stabilized gold nanoparticle interactions.

    Eichmann, Shannon L; Bevan, Michael A

    2010-09-21

    We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media. Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects. Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials. Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with predictions. These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods.

  5. Coarse-grained versus atomistic simulations : realistic interaction free energies for real proteins

    May, Ali; Pool, René; van Dijk, Erik; Bijlard, Jochem; Abeln, Sanne; Heringa, Jaap; Feenstra, K Anton

    2014-01-01

    MOTIVATION: To assess whether two proteins will interact under physiological conditions, information on the interaction free energy is needed. Statistical learning techniques and docking methods for predicting protein-protein interactions cannot quantitatively estimate binding free energies. Full

  6. Coarse-grained versus atomistic simulations: realistic interaction free energies for real proteins

    May, A.; Pool, R.; van Dijk, E.; Bijlard, J.; Abeln, S.; Heringa, J.; Feenstra, K.A.

    2014-01-01

    MOTIVATION: To assess whether two proteins will interact under physiological conditions, information on the interaction free energy is needed. Statistical learning techniques and docking methods for predicting protein-protein interactions cannot quantitatively estimate binding free energies. Full

  7. Exploration of the omics evidence landscape: adding qualitative labels to predicted protein-protein interactions

    Noort, V. van; Snel, B.; Huynen, M.A.

    2007-01-01

    ABSTRACT: BACKGROUND: In the post-genomic era various functional genomics, proteomics and computational techniques have been developed to elucidate the protein interaction network. While some of these techniques are specific for a certain type of interaction, most predict a mixture of interactions.

  8. Exploration of the omics evidence landscape: adding qualitative labels to predicted protein-protein interactions.

    Noort, V. van; Snel, B.; Huynen, M.A.

    2007-01-01

    BACKGROUND: In the post-genomic era various functional genomics, proteomics and computational techniques have been developed to elucidate the protein interaction network. While some of these techniques are specific for a certain type of interaction, most predict a mixture of interactions.

  9. Interaction of influenza virus proteins with nucleosomes

    Garcia-Robles, Inmaculada; Akarsu, Hatice; Mueller, Christoph W.; Ruigrok, Rob W.H.; Baudin, Florence

    2005-01-01

    During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed

  10. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg

    2006-01-01

    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

  11. New roles of the human Suv3 helicase in genome maintenance

    Venø, Susanne Trillingsgaard

    During her PhD studies, Susanne Trillingsgaard Venø carried out research into the role of the human Suv3 protein in stabilising the human genome – DNA. Suv3 is a helicase that separates the two strands of the DNA’s double helix. Throughout our lives, the DNA in our cells is constantly exposed...... maintenance. Based on these new research results, the Suv3 protein could be a valuable model for genome stability as an important factor in our understanding of why we get old....

  12. Prediction of Protein–Protein Interactions by Evidence Combining Methods

    Ji-Wei Chang

    2016-11-01

    Full Text Available Most cellular functions involve proteins’ features based on their physical interactions with other partner proteins. Sketching a map of protein–protein interactions (PPIs is therefore an important inception step towards understanding the basics of cell functions. Several experimental techniques operating in vivo or in vitro have made significant contributions to screening a large number of protein interaction partners, especially high-throughput experimental methods. However, computational approaches for PPI predication supported by rapid accumulation of data generated from experimental techniques, 3D structure definitions, and genome sequencing have boosted the map sketching of PPIs. In this review, we shed light on in silico PPI prediction methods that integrate evidence from multiple sources, including evolutionary relationship, function annotation, sequence/structure features, network topology and text mining. These methods are developed for integration of multi-dimensional evidence, for designing the strategies to predict novel interactions, and for making the results consistent with the increase of prediction coverage and accuracy.

  13. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

    Albrecht von Brunn

    2007-05-01

    Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

  14. Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

    The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

  15. Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

    Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong; Luo, Zhao-Qing; Shen, Xihui

    2014-05-20

    Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells

  16. Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen.

    Plante, Geneviève; Lusignan, Marie-France; Lafleur, Michel; Manjunath, Puttaswamy

    2015-08-15

    Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

  17. Non-B DNA Secondary Structures and Their Resolution by RecQ Helicases

    Sudha Sharma

    2011-01-01

    Full Text Available In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics.

  18. RNases and Helicases in Gram-Positive Bacteria.

    Durand, Sylvain; Condon, Ciaran

    2018-04-01

    RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis , the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis , the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.

  19. Towards a map of the Populus biomass protein-protein interaction network

    Beers, Eric [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Brunner, Amy [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Helm, Richard [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Dickerman, Allan [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)

    2015-07-31

    Biofuels can be produced from a variety of plant feedstocks. The value of a particular feedstock for biofuels production depends in part on the degree of difficulty associated with the extraction of fermentable sugars from the plant biomass. The wood of trees is potentially a rich source fermentable sugars. However, the sugars in wood exist in a tightly cross-linked matrix of cellulose, hemicellulose, and lignin, making them largely recalcitrant to release and fermentation for biofuels production. Before breeders and genetic engineers can effectively develop plants with reduced recalcitrance to fermentation, it is necessary to gain a better understanding of the fundamental biology of the mechanisms responsible for wood formation. Regulatory, structural, and enzymatic proteins are required for the complicated process of wood formation. To function properly, proteins must interact with other proteins. Yet, very few of the protein-protein interactions necessary for wood formation are known. The main objectives of this project were to 1) identify new protein-protein interactions relevant to wood formation, and 2) perform in-depth characterizations of selected protein-protein interactions. To identify relevant protein-protein interactions, we cloned a set of approximately 400 genes that were highly expressed in the wood-forming tissue (known as secondary xylem) of poplar (Populus trichocarpa). We tested whether the proteins encoded by these biomass genes interacted with each other in a binary matrix design using the yeast two-hybrid (Y2H) method for protein-protein interaction discovery. We also tested a subset of the 400 biomass proteins for interactions with all proteins present in wood-forming tissue of poplar in a biomass library screen design using Y2H. Together, these two Y2H screens yielded over 270 interactions involving over 75 biomass proteins. For the second main objective we selected several interacting pairs or groups of interacting proteins for in

  20. Protein-Protein Interactions Prediction Based on Iterative Clique Extension with Gene Ontology Filtering

    Lei Yang

    2014-01-01

    Full Text Available 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.

  1. Using the clustered circular layout as an informative method for visualizing protein-protein interaction networks.

    Fung, David C Y; Wilkins, Marc R; Hart, David; Hong, Seok-Hee

    2010-07-01

    The force-directed layout is commonly used in computer-generated visualizations of protein-protein interaction networks. While it is good for providing a visual outline of the protein complexes and their interactions, it has two limitations when used as a visual analysis method. The first is poor reproducibility. Repeated running of the algorithm does not necessarily generate the same layout, therefore, demanding cognitive readaptation on the investigator's part. The second limitation is that it does not explicitly display complementary biological information, e.g. Gene Ontology, other than the protein names or gene symbols. Here, we present an alternative layout called the clustered circular layout. Using the human DNA replication protein-protein interaction network as a case study, we compared the two network layouts for their merits and limitations in supporting visual analysis.

  2. Emergence of modularity and disassortativity in protein-protein interaction networks.

    Wan, Xi; Cai, Shuiming; Zhou, Jin; Liu, Zengrong

    2010-12-01

    In this paper, we present a simple evolution model of protein-protein interaction networks by introducing a rule of small-preference duplication of a node, meaning that the probability of a node chosen to duplicate is inversely proportional to its degree, and subsequent divergence plus nonuniform heterodimerization based on some plausible mechanisms in biology. We show that our model cannot only reproduce scale-free connectivity and small-world pattern, but also exhibit hierarchical modularity and disassortativity. After comparing the features of our model with those of real protein-protein interaction networks, we believe that our model can provide relevant insights into the mechanism underlying the evolution of protein-protein interaction networks. © 2010 American Institute of Physics.

  3. In silico modeling of the yeast protein and protein family interaction network

    Goh, K.-I.; Kahng, B.; Kim, D.

    2004-03-01

    Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

  4. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

    Ji'an Pan

    Full Text Available Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12 provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

  5. Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

    Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

    2016-09-01

    In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  6. DiffSLC: A graph centrality method to detect essential proteins of a protein-protein interaction network.

    Mistry, Divya; Wise, Roger P; Dickerson, Julie A

    2017-01-01

    Identification of central genes and proteins in biomolecular networks provides credible candidates for pathway analysis, functional analysis, and essentiality prediction. The DiffSLC centrality measure predicts central and essential genes and proteins using a protein-protein interaction network. Network centrality measures prioritize nodes and edges based on their importance to the network topology. These measures helped identify critical genes and proteins in biomolecular networks. The proposed centrality measure, DiffSLC, combines the number of interactions of a protein and the gene coexpression values of genes from which those proteins were translated, as a weighting factor to bias the identification of essential proteins in a protein interaction network. Potentially essential proteins with low node degree are promoted through eigenvector centrality. Thus, the gene coexpression values are used in conjunction with the eigenvector of the network's adjacency matrix and edge clustering coefficient to improve essentiality prediction. The outcome of this prediction is shown using three variations: (1) inclusion or exclusion of gene co-expression data, (2) impact of different coexpression measures, and (3) impact of different gene expression data sets. For a total of seven networks, DiffSLC is compared to other centrality measures using Saccharomyces cerevisiae protein interaction networks and gene expression data. Comparisons are also performed for the top ranked proteins against the known essential genes from the Saccharomyces Gene Deletion Project, which show that DiffSLC detects more essential proteins and has a higher area under the ROC curve than other compared methods. This makes DiffSLC a stronger alternative to other centrality methods for detecting essential genes using a protein-protein interaction network that obeys centrality-lethality principle. DiffSLC is implemented using the igraph package in R, and networkx package in Python. The python package can be

  7. Yeast Interacting Proteins Database: YNL152W, YMR032W [Yeast Interacting Proteins Database

    Full Text Available YNL152W INN1 Essential protein that associates with the contractile actomyosin ring... Bait description Essential protein that associates with the contractile actomyosin ring, required for ingre

  8. Yeast Interacting Proteins Database: YNL258C, YGL145W [Yeast Interacting Proteins Database

    Full Text Available YNL258C DSL1 Peripheral membrane protein required for Golgi-to-ER retrograde traffi...t description Peripheral membrane protein required for Golgi-to-ER retrograde traffic; component of the ER t

  9. Yeast Interacting Proteins Database: YNL216W, YLR453C [Yeast Interacting Proteins Database

    Full Text Available YNL216W RAP1 DNA-binding protein involved in either activation or repression of transcription, depending...NA-binding protein involved in either activation or repression of transcription, depending on binding site c

  10. Yeast Interacting Proteins Database: YOL006C, YMR233W [Yeast Interacting Proteins Database

    Full Text Available fusion protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey as prey (1) Rows with t...on protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey

  11. Yeast Interacting Proteins Database: YKL002W, YFL034C-B [Yeast Interacting Proteins Database

    Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes...ntegral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesi

  12. Yeast Interacting Proteins Database: YJR091C, YKL002W [Yeast Interacting Proteins Database

    Full Text Available g of integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly sy... integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthe

  13. Yeast Interacting Proteins Database: YCL046W, YGL115W [Yeast Interacting Proteins Database

    Full Text Available YCL046W - Dubious open reading frame unlikely to encode a protein, based on availab...ading frame unlikely to encode a protein, based on available experimental and comparative sequence data; par

  14. Influenza A Virus-Host Protein Interactions Control Viral Pathogenesis.

    Zhao, Mengmeng; Wang, Lingyan; Li, Shitao

    2017-08-01

    The influenza A virus (IAV), a member of the Orthomyxoviridae family, is a highly transmissible respiratory pathogen and represents a continued threat to global health with considerable economic and social impact. IAV is a zoonotic virus that comprises a plethora of strains with different pathogenic profiles. The different outcomes of viral pathogenesis are dependent on the engagement between the virus and the host cellular protein interaction network. The interactions may facilitate virus hijacking of host molecular machinery to fulfill the viral life cycle or trigger host immune defense to eliminate the virus. In recent years, much effort has been made to discover the virus-host protein interactions and understand the underlying mechanisms. In this paper, we review the recent advances in our understanding of IAV-host interactions and how these interactions contribute to host defense and viral pathogenesis.

  15. Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

    Szewczyk, Maciej; Fedoryszak-Kuśka, Natalia; Tkaczuk, Katarzyna; Dobrucki, Jurek; Waligórska, Agnieszka; Stępień, Piotr P

    2017-01-01

    The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

  16. In vivo interactions between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA dependent polymerase VP1

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  17. Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent polymerase, VP1

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  18. Energetics of the protein-DNA-water interaction

    Marabotti Anna

    2007-01-01

    Full Text Available Abstract Background To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species. Results An initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts. Conclusion This study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation mostly cancel.

  19. The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance.

    Amit Dipak Amin

    2010-11-01

    Full Text Available The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout

  20. MPact: the MIPS protein interaction resource on yeast.

    Güldener, Ulrich; Münsterkötter, Martin; Oesterheld, Matthias; Pagel, Philipp; Ruepp, Andreas; Mewes, Hans-Werner; Stümpflen, Volker

    2006-01-01

    In recent years, the Munich Information Center for Protein Sequences (MIPS) yeast protein-protein interaction (PPI) dataset has been used in numerous analyses of protein networks and has been called a gold standard because of its quality and comprehensiveness [H. Yu, N. M. Luscombe, H. X. Lu, X. Zhu, Y. Xia, J. D. Han, N. Bertin, S. Chung, M. Vidal and M. Gerstein (2004) Genome Res., 14, 1107-1118]. MPact and the yeast protein localization catalog provide information related to the proximity of proteins in yeast. Beside the integration of high-throughput data, information about experimental evidence for PPIs in the literature was compiled by experts adding up to 4300 distinct PPIs connecting 1500 proteins in yeast. As the interaction data is a complementary part of CYGD, interactive mapping of data on other integrated data types such as the functional classification catalog [A. Ruepp, A. Zollner, D. Maier, K. Albermann, J. Hani, M. Mokrejs, I. Tetko, U. Güldener, G. Mannhaupt, M. Münsterkötter and H. W. Mewes (2004) Nucleic Acids Res., 32, 5539-5545] is possible. A survey of signaling proteins and comparison with pathway data from KEGG demonstrates that based on these manually annotated data only an extensive overview of the complexity of this functional network can be obtained in yeast. The implementation of a web-based PPI-analysis tool allows analysis and visualization of protein interaction networks and facilitates integration of our curated data with high-throughput datasets. The complete dataset as well as user-defined sub-networks can be retrieved easily in the standardized PSI-MI format. The resource can be accessed through http://mips.gsf.de/genre/proj/mpact.

  1. Prediction of thermodynamic instabilities of protein solutions from simple protein–protein interactions

    D’Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio

    2013-01-01

    Highlights: ► We propose a model of effective protein–protein interaction embedding solvent effects. ► A previous square-well model is enhanced by giving to the interaction a free energy character. ► The temperature dependence of the interaction is due to entropic effects of the solvent. ► The validity of the original SW model is extended to entropy driven phase transitions. ► We get good fits for lysozyme and haemoglobin spinodal data taken from literature. - Abstract: Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein–protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential

  2. An analysis pipeline for the inference of protein-protein interaction networks

    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.

  3. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  4. A new protein-protein interaction sensor based on tripartite split-GFP association.

    Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-10-04

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

  5. Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

    Chin-Jen Wu

    2013-06-01

    Full Text Available 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.

  6. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    Jillian L Blatti

    Full Text Available 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.

  7. Dissection of the functional domains of an archaeal holliday junction helicase

    Hong, Ye; Chu, Mingzhu; Li, Yansheng

    2012-01-01

    Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from...... conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting...

  8. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  9. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    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

  10. Interaction of milk whey protein with common phenolic acids

    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.

  11. Proteins that interact with GTP during sporulation of Bacillus subtilis

    Mitchell, C.; Vary, J.C.

    1989-01-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [α- 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 [α- 32 P]GTP or [γ- 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

  12. Dynamics of DNA conformations and DNA-protein interaction

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

    2005-01-01

    Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

  13. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  14. Analytical techniques for the study of polyphenol-protein interactions.

    Poklar Ulrih, Nataša

    2017-07-03

    This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol-protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol-protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol-protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol-protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.

  15. BioPlex Display: An Interactive Suite for Large-Scale AP-MS Protein-Protein Interaction Data.

    Schweppe, Devin K; Huttlin, Edward L; Harper, J Wade; Gygi, Steven P

    2018-01-05

    The development of large-scale data sets requires a new means to display and disseminate research studies to large audiences. Knowledge of protein-protein interaction (PPI) networks has become a principle interest of many groups within the field of proteomics. At the confluence of technologies, such as cross-linking mass spectrometry, yeast two-hybrid, protein cofractionation, and affinity purification mass spectrometry (AP-MS), detection of PPIs can uncover novel biological inferences at a high-throughput. Thus new platforms to provide community access to large data sets are necessary. To this end, we have developed a web application that enables exploration and dissemination of the growing BioPlex interaction network. BioPlex is a large-scale interactome data set based on AP-MS of baits from the human ORFeome. The latest BioPlex data set release (BioPlex 2.0) contains 56 553 interactions from 5891 AP-MS experiments. To improve community access to this vast compendium of interactions, we developed BioPlex Display, which integrates individual protein querying, access to empirical data, and on-the-fly annotation of networks within an easy-to-use and mobile web application. BioPlex Display enables rapid acquisition of data from BioPlex and development of hypotheses based on protein interactions.

  16. Yeast Interacting Proteins Database: YLR347C, YLR377C [Yeast Interacting Proteins Database

    Full Text Available gy-mediated degradation depending on growth conditions; interacts with Vid30p Rows with this prey as prey (4...r autophagy-mediated degradation depending on growth conditions; interacts with V

  17. Yeast Interacting Proteins Database: YNL189W, YLR377C [Yeast Interacting Proteins Database

    Full Text Available phagy-mediated degradation depending on growth conditions; interacts with Vid30p Rows with this prey as prey...ated or autophagy-mediated degradation depending on growth conditions; interacts

  18. Yeast Interacting Proteins Database: YML064C, YLR377C [Yeast Interacting Proteins Database

    Full Text Available d or autophagy-mediated degradation depending on growth conditions; interacts with Vid30p Rows with this pre...er proteasome-mediated or autophagy-mediated degradation depending on growth conditions; interacts with Vid3

  19. Atom depth analysis delineates mechanisms of protein intermolecular interactions

    Alocci, Davide; Bernini, Andrea; Niccolai, Neri

    2013-01-01

    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

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

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

    2009-01-01

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

  1. Interactions of polyphenols with carbohydrates, lipids and proteins.

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Studying RNA-protein interactions in vivo by RNA immunoprecipitation

    Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

    2011-01-01

    and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

  3. Systematic discovery of new recognition peptides mediating protein interaction networks

    Neduva, Victor; Linding, Rune; Su-Angrand, Isabelle

    2005-01-01

    Many aspects of cell signalling, trafficking, and targeting are governed by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or "linear motif" (e.g., SH3 binding to PxxP). Many domains...... by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or "linear motif" (e.g., SH3 binding to PxxP). Many domains are known, though comparatively few linear motifs have been discovered. Their short length...

  4. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  5. Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

    Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

    2017-06-01

    Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

  6. C2 Domains as Protein-Protein Interaction Modules in the Ciliary Transition Zone

    Kim Remans

    2014-07-01

    Full Text Available RPGR-interacting protein 1 (RPGRIP1 is mutated in the eye disease Leber congenital amaurosis (LCA and its structural homolog, RPGRIP1-like (RPGRIP1L, is mutated in many different ciliopathies. Both are multidomain proteins that are predicted to interact with retinitis pigmentosa G-protein regulator (RPGR. RPGR is mutated in X-linked retinitis pigmentosa and is located in photoreceptors and primary cilia. We solved the crystal structure of the complex between the RPGR-interacting domain (RID of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR similarly. RPGRIP1 binding to RPGR affects the interaction with PDEδ, the cargo shuttling factor for prenylated ciliary proteins. RPGRIP1-RID is a C2 domain with a canonical β sandwich structure that does not bind Ca2+ and/or phospholipids and thus constitutes a unique type of protein-protein interaction module. Judging from the large number of C2 domains in most of the ciliary transition zone proteins identified thus far, the structure presented here seems to constitute a cilia-specific module that is present in multiprotein transition zone complexes.

  7. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    Eric A Yen

    2014-05-01

    Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

  8. A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

    Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  9. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  10. Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

    Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

    2017-12-01

    RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

  11. Dough and hearth bread characteristics influenced by protein composition, protein content, DATEM, and their interactions

    Aamodt, A.; Magnus, E.M.; Hollung, K.; Uhlen, A.K.; Færgestad, E.M.

    2005-01-01

    The effects of protein composition, protein content, diacetyl tartaric acid ester of monoglycerides (DATEM), and their interaction on dough and bread characteristics were studied by small- and pilot-scale hearth bread baking, dough rheological testing using the Kieffer extensibility rig, and size

  12. Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle

    Nora López

    2012-09-01

    Full Text Available The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article.

  13. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    Jaume Torres

    2015-06-01

    Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  14. Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

    Hu, Xiaotang; Li, Hongbin

    2014-10-01

    Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Cold-set globular protein gels: Interactions, structure and rheology as a function of protein concentration.

    Alting, A.C.; Hamer, R.J.; Kruif, de C.G.

    2003-01-01

    We identified the contribution of covalent and noncovalent interactions to the scaling behavior of the structural and rheological properties in a cold gelling protein system. The system we studied consisted of two types of whey protein aggregates, equal in size but different in the amount of

  16. Identification of proteins interacting with Arabidopsis ACD11

    Petersen, Nikolaj H T; Joensen, Jan; McKinney, Lea V

    2009-01-01

    The Arabidopsis ACD11 gene encodes a sphingosine transfer protein and was identified by the accelerated cell death phenotype of the loss of function acd11 mutant, which exhibits heightened expression of genes involved in the disease resistance hypersensitive response (HR). We used ACD11 as bait...... in a yeast two-hybrid screen of an Arabidopsis cDNA library to identify ACD11 interacting proteins. One interactor identified is a protein of unknown function with an RNA recognition motif (RRM) designated BPA1 (binding partner of ACD11). Co-immunoprecipitation experiments confirmed the ACD11-BPA1...

  17. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  18. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    Physics approaches focus on uncovering, modeling and quantitating the general principles governing the micro and macro universe. This has always been an important component of biological research, however recent advances in experimental techniques and the accumulation of unprecedented genome-scale experimental data produced by these novel technologies now allow for addressing fundamental questions on a large scale. These relate to molecular interactions, principles of bimolecular recognition, and mechanisms of signal propagation. The functioning of a cell requires a variety of intermolecular interactions including protein-protein, protein-DNA, protein-RNA, hormones, peptides, small molecules, lipids and more. Biomolecules work together to provide specific functions and perturbations in intermolecular communication channels often lead to cellular malfunction and disease. A full understanding of the interactome requires an in-depth grasp of the biophysical principles underlying individual interactions as well as their organization in cellular networks. Phenomena can be described at different levels of abstraction. Computational and systems biology strive to model cellular processes by integrating and analyzing complex data from multiple experimental sources using interdisciplinary tools. As a result, both the causal relationships between the variables and the general features of the system can be discovered, which even without knowing the details of the underlying mechanisms allow for putting forth hypotheses and predicting the behavior of the systems in response to perturbation. And here lies the strength of in silico models which provide control and predictive power. At the same time, the complexity of individual elements and molecules can be addressed by the fields of molecular biophysics, physical biology and structural biology, which focus on the underlying physico-chemical principles and may explain the molecular mechanisms of cellular function. In this issue

  19. The role of exon shuffling in shaping protein-protein interaction networks

    França Gustavo S

    2010-12-01

    Full Text Available Abstract Background Physical protein-protein interaction (PPI is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of PPIs are the result of domain-domain interactions. Exon shuffling, intron-mediated recombination of exons from existing genes, is known to have been a major mechanism of domain shuffling in metazoans. Thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of PPI networks. Results We tested our hypothesis by compiling exon shuffling and PPI data from six eukaryotic species: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Cryptococcus neoformans and Arabidopsis thaliana. For all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree (number of interacting partners in PPI networks. Furthermore, we verified that a set of protein domains that are simultaneously promiscuous (known to interact to multiple types of other domains, self-interacting (able to interact with another copy of themselves and abundant in the genomes presents a stronger signal for exon shuffling. Conclusions Exon shuffling appears to have been a recurrent mechanism for the emergence of new PPIs along metazoan evolution. In metazoan genomes, exon shuffling also promoted the expansion of some protein domains. We speculate that their promiscuous and self-interacting properties may have been decisive for that expansion.

  20. Study of the interactions between riboflavin and protein

    Zhao Hongwei; Zhang Zhaoxia; Zhu Hongping; Ge Min; Miao Jinling; Wang Wenfeng; Yao Side

    2006-01-01

    Riboflavin (VB 2 ), a vitamin that is a natural constituent of living organisms, plays an important role in the process of metabolism and it is essential for normal cellular functions and growth. As an endogenous photosensitizer, riboflavin induces photooxidation damages to cells of skin and eye, causing inflammation, accelerated aging and mutation. The photodynamic actions of riboflavin on DNA have been studied extensively, however, its photodynamic actions on protein and enzyme are less studied due to the complex types and structures of proteins, and less attentions have been paid to photosensitive protein damages than DNA. In our lab, the interactions between riboflavin and serum albumin and lysozyme have been carried out by using transient absorption spectrometry, emission spectrometer analysis and electrophoresis techniques. It was found that stable state products and transient process of photosensitive damage to proteins were closely relative to concentration of riboflavin, time of irradiation and the atmosphere of the solutions. The results indicates that proteins can be photosensitive damaged by riboflavin irradiated by UV lights. Riboflavin can quench the intrinsic fluorescence of protein. Both dynamic and static quenching are simultaneous involved. The binding constants, the kinetics and spectroscopic properties of riboflavin interaction with albumin and lysozyme have also been investigated. Mechanisms of the photosensitive damages to proteins were discussed. The experiments also indicated that antioxidants such as melatonin and chlorogenic acid can reduce the damage of lysozyme effectively. (authors)

  1. Protein solubility and folding enhancement by interaction with RNA.

    Seong Il Choi

    Full Text Available While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.

  2. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  3. Transcriptional robustness and protein interactions are associated in yeast

    Conant Gavin C

    2011-05-01

    Full Text Available Abstract Background Robustness to insults, both external and internal, is a characteristic feature of life. One level of biological organization for which noise and robustness have been extensively studied is gene expression. Cells have a variety of mechanisms for buffering noise in gene expression, but it is not completely clear what rules govern whether or not a given gene uses such tools to maintain appropriate expression. Results Here, we show a general association between the degree to which yeast cells have evolved mechanisms to buffer changes in gene expression and whether they possess protein-protein interactions. We argue that this effect bears an affinity to epistasis, because yeast appears to have evolved regulatory mechanisms such that distant changes in gene copy number for a protein-protein interaction partner gene can alter a gene's expression. This association is not unexpected given recent work linking epistasis and the deleterious effects of changes in gene dosage (i.e., the dosage balance hypothesis. Using gene expression data from artificial aneuploid strains of bakers' yeast, we found that genes coding for proteins that physically interact with other proteins show less expression variation in response to aneuploidy than do other genes. This effect is even more pronounced for genes whose products interact with proteins encoded on aneuploid chromosomes. We further found that genes targeted by transcription factors encoded on aneuploid chromosomes were more likely to change in expression after aneuploidy. Conclusions We suggest that these observations can be best understood as resulting from the higher fitness cost of misexpression in epistatic genes and a commensurate greater regulatory control of them.

  4. Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

    Linda S Kaltenbach

    2007-05-01

    Full Text Available Huntington's disease (HD is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to

  5. Efficient extraction of protein-protein interactions from full-text articles.

    Hakenberg, Jörg; Leaman, Robert; Vo, Nguyen Ha; Jonnalagadda, Siddhartha; Sullivan, Ryan; Miller, Christopher; Tari, Luis; Baral, Chitta; Gonzalez, Graciela

    2010-01-01

    Proteins and their interactions govern virtually all cellular processes, such as regulation, signaling, metabolism, and structure. Most experimental findings pertaining to such interactions are discussed in research papers, which, in turn, get curated by protein interaction databases. Authors, editors, and publishers benefit from efforts to alleviate the tasks of searching for relevant papers, evidence for physical interactions, and proper identifiers for each protein involved. The BioCreative II.5 community challenge addressed these tasks in a competition-style assessment to evaluate and compare different methodologies, to make aware of the increasing accuracy of automated methods, and to guide future implementations. In this paper, we present our approaches for protein-named entity recognition, including normalization, and for extraction of protein-protein interactions from full text. Our overall goal is to identify efficient individual components, and we compare various compositions to handle a single full-text article in between 10 seconds and 2 minutes. We propose strategies to transfer document-level annotations to the sentence-level, which allows for the creation of a more fine-grained training corpus; we use this corpus to automatically derive around 5,000 patterns. We rank sentences by relevance to the task of finding novel interactions with physical evidence, using a sentence classifier built from this training corpus. Heuristics for paraphrasing sentences help to further remove unnecessary information that might interfere with patterns, such as additional adjectives, clauses, or bracketed expressions. In BioCreative II.5, we achieved an f-score of 22 percent for finding protein interactions, and 43 percent for mapping proteins to UniProt IDs; disregarding species, f-scores are 30 percent and 55 percent, respectively. On average, our best-performing setup required around 2 minutes per full text. All data and pattern sets as well as Java classes that

  6. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Tuteja, Narendra; Banu, Mst Sufara Akhter; Huda, Kazi Md Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  7. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Narendra Tuteja

    Full Text Available The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum and its novel function in salinity stress tolerance in plant.The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities.To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  8. Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

    Andersen, Tonni Grube; Nintemann, Sebastian; Marek, Magdalena

    2016-01-01

    When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true-from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased se...

  9. Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein-Protein Interaction.

    Banda, Srikanth; Cao, Nan; Tse-Dinh, Yuk-Ching

    2017-09-15

    We report here a distinct mechanism of interaction between topoisomerase I and RNA polymerase in Mycobacterium tuberculosis and Mycobacterium smegmatis that has evolved independently from the previously characterized interaction between bacterial topoisomerase I and RNA polymerase. Bacterial DNA topoisomerase I is responsible for preventing the hyper-negative supercoiling of genomic DNA. The association of topoisomerase I with RNA polymerase during transcription elongation could efficiently relieve transcription-driven negative supercoiling. Our results demonstrate a direct physical interaction between the C-terminal domains of topoisomerase I (TopoI-CTDs) and the β' subunit of RNA polymerase of M. smegmatis in the absence of DNA. The TopoI-CTDs in mycobacteria are evolutionarily unrelated in amino acid sequence and three-dimensional structure to the TopoI-CTD found in the majority of bacterial species outside Actinobacteria, including Escherichia coli. The functional interaction between topoisomerase I and RNA polymerase has evolved independently in mycobacteria and E. coli, with distinctively different structural elements of TopoI-CTD utilized for this protein-protein interaction. Zinc ribbon motifs in E. coli TopoI-CTD are involved in the interaction with RNA polymerase. For M. smegmatis TopoI-CTD, a 27-amino-acid tail that is rich in basic residues at the C-terminal end is responsible for the interaction with RNA polymerase. Overexpression of recombinant TopoI-CTD in M. smegmatis competed with the endogenous topoisomerase I for protein-protein interactions with RNA polymerase. The TopoI-CTD overexpression resulted in decreased survival following treatment with antibiotics and hydrogen peroxide, supporting the importance of the protein-protein interaction between topoisomerase I and RNA polymerase during stress response of mycobacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. False positive reduction in protein-protein interaction predictions using gene ontology annotations

    Lin Yen-Han

    2007-07-01

    Full Text Available Abstract Background Many crucial cellular operations such as metabolism, signalling, and regulations are based on protein-protein interactions. However, the lack of robust protein-protein interaction information is a challenge. One reason for the lack of solid protein-protein interaction information is poor agreement between experimental findings and computational sets that, in turn, comes from huge false positive predictions in computational approaches. Reduction of false positive predictions and enhancing true positive fraction of computationally predicted protein-protein interaction datasets based on highly confident experimental results has not been adequately investigated. Results Gene Ontology (GO annotations were used to reduce false positive protein-protein interactions (PPI pairs resulting from computational predictions. Using experimentally obtained PPI pairs as a training dataset, eight top-ranking keywords were extracted from GO molecular function annotations. The sensitivity of these keywords is 64.21% in the yeast experimental dataset and 80.83% in the worm experimental dataset. The specificities, a measure of recovery power, of these keywords applied to four predicted PPI datasets for each studied organisms, are 48.32% and 46.49% (by average of four datasets in yeast and worm, respectively. Based on eight top-ranking keywords and co-localization of interacting proteins a set of two knowledge rules were deduced and applied to remove false positive protein pairs. The 'strength', a measure of improvement provided by the rules was defined based on the signal-to-noise ratio and implemented to measure the applicability of knowledge rules applying to the predicted PPI datasets. Depending on the employed PPI-predicting methods, the strength varies between two and ten-fold of randomly removing protein pairs from the datasets. Conclusion Gene Ontology annotations along with the deduced knowledge rules could be implemented to partially

  11. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-04-22

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  12. Boosting compound-protein interaction prediction by deep learning.

    Tian, Kai; Shao, Mingyu; Wang, Yang; Guan, Jihong; Zhou, Shuigeng

    2016-11-01

    The identification of interactions between compounds and proteins plays an important role in network pharmacology and drug discovery. However, experimentally identifying compound-protein interactions (CPIs) is generally expensive and time-consuming, computational approaches are thus introduced. Among these, machine-learning based methods have achieved a considerable success. However, due to the nonlinear and imbalanced nature of biological data, many machine learning approaches have their own limitations. Recently, deep learning techniques show advantages over many state-of-the-art machine learning methods in some applications. In this study, we aim at improving the performance of CPI prediction based on deep learning, and propose a method called DL-CPI (the abbreviation of Deep Learning for Compound-Protein Interactions prediction), which employs deep neural network (DNN) to effectively learn the representations of compound-protein pairs. Extensive experiments show that DL-CPI can learn useful features of compound-protein pairs by a layerwise abstraction, and thus achieves better prediction performance than existing methods on both balanced and imbalanced datasets. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Connecting Protein Structure to Intermolecular Interactions: A Computer Modeling Laboratory

    Abualia, Mohammed; Schroeder, Lianne; Garcia, Megan; Daubenmire, Patrick L.; Wink, Donald J.; Clark, Ginevra A.

    2016-01-01

    An understanding of protein folding relies on a solid foundation of a number of critical chemical concepts, such as molecular structure, intra-/intermolecular interactions, and relating structure to function. Recent reports show that students struggle on all levels to achieve these understandings and use them in meaningful ways. Further, several…

  14. Screening of cellular proteins that interact with the classical swine ...

    In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database ...

  15. Screening of cellular proteins that interact with the classical swine ...

    2014-01-27

    Jan 27, 2014 ... to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell. (SUVEC). Alignment ... development. The finding of ..... were unknown, the results of the BLAST against the human.

  16. Diverse role of CBL-interacting protein kinases in plant

    admin

    Diverse role of CBL-interacting protein kinases in plant. Most of the extracellular and ... to their role in stress signalling. Their role in transport of plant hormone auxin and mechanism of action in stress response shed new light on diverse role of.

  17. Protein and Drug Interactions in the Minor Groove of DNA

    Morávek, Z.; Neidle, S.; Schneider, Bohdan

    2002-01-01

    Roč. 30, č. 5 (2002), s. 1182-1191 ISSN 0305-1048 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : protein * DNA * interactions Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 7.051, year: 2002

  18. Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression

    Vandepoele Klaas

    2009-06-01

    Full Text Available Abstract Background Large-scale identification of the interrelationships between different components of the cell, such as the interactions between proteins, has recently gained great interest. However, unraveling large-scale protein-protein interaction maps is laborious and expensive. Moreover, assessing the reliability of the interactions can be cumbersome. Results In this study, we have developed a computational method that exploits the existing knowledge on protein-protein interactions in diverse species through orthologous relations on the one hand, and functional association data on the other hand to predict and filter protein-protein interactions in Arabidopsis thaliana. A highly reliable set of protein-protein interactions is predicted through this integrative approach making use of existing protein-protein interaction data from yeast, human, C. elegans and D. melanogaster. Localization, biological process, and co-expression data are used as powerful indicators for protein-protein interactions. The functional repertoire of the identified interactome reveals interactions between proteins functioning in well-conserved as well as plant-specific biological processes. We observe that although common mechanisms (e.g. actin polymerization and components (e.g. ARPs, actin-related proteins exist between different lineages, they are active in specific processes such as growth, cancer metastasis and trichome development in yeast, human and Arabidopsis, respectively. Conclusion We conclude that the integration of orthology with functional association data is adequate to predict protein-protein interactions. Through this approach, a high number of novel protein-protein interactions with diverse biological roles is discovered. Overall, we have predicted a reliable set of protein-protein interactions suitable for further computational as well as experimental analyses.

  19. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    Haidar, Malak

    2017-09-08

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

  20. Yeast as a model system to study RecQ helicase function

    Ashton, Thomas M; Hickson, Ian David

    2010-01-01

    Mutations in the highly conserved RecQ helicase, BLM, cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1(+), respectively. Studies in these yeast species have revealed a plethora...... of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast...

  1. Towards a better understanding of the specificity of protein-protein interaction

    Kysilka, Jiří; Vondrášek, Jiří

    2012-01-01

    Roč. 25, č. 11 (2012), s. 604-615 ISSN 0952-3499 R&D Projects: GA ČR GAP208/10/0725; GA ČR GAP302/10/0427; GA MŠk(CZ) LH11020 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : protein-protein interaction * molecular recognition * x-ray structure analysis * empirical potentials * side chain-side chain interaction * interaction energy * bioinformatics Subject RIV: CE - Biochemistry Impact factor: 3.006, year: 2012

  2. Probing hydrogen bonding interactions and proton transfer in proteins

    Nie, Beining

    Scope and method of study. Hydrogen bonding is a fundamental element in protein structure and function. Breaking a single hydrogen bond may impair the stability of a protein. It is therefore important to probe dynamic changes in hydrogen bonding interactions during protein folding and function. Time-resolved Fourier transform infrared spectroscopy is highly sensitive to hydrogen bonding interactions. However, it lacks quantitative correlation between the vibrational frequencies and the number, type, and strength of hydrogen bonding interactions of ionizable and polar residues. We employ quantum physics theory based ab initio calculations to study the effects of hydrogen bonding interactions on vibrational frequencies of Asp, Glu, and Tyr residues and to develop vibrational spectral markers for probing hydrogen bonding interactions using infrared spectroscopy. In addition, proton transfer process plays a crucial role in a wide range of energy transduction, signal transduction, and enzymatic reactions. We study the structural basis for proton transfer using photoactive yellow protein as an excellent model system. Molecular dynamics simulation is employed to investigate the structures of early intermediate states. Quantum theory based ab initio calculations are used to study the impact of hydrogen bond interactions on proton affinity and proton transfer. Findings and conclusions. Our extensive density function theory based calculations provide rich structural, spectral, and energetic information on hydrogen bonding properties of protonated side chain groups of Asp/Glu and Tyr. We developed vibrational spectral markers and 2D FTIR spectroscopy for structural characterization on the number and the type of hydrogen bonding interactions of the COOH group of Asp/Glu and neutral phenolic group of Tyr. These developments greatly enhance the power of time-resolved FTIR spectroscopy as a major experimental tool for structural characterization of functionally important

  3. Prediction of protein-protein interaction sites in sequences and 3D structures by random forests.

    Mile Sikić

    2009-01-01

    Full Text Available Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i a combination of sequence- and structure-derived parameters and (ii sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras-Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.

  4. Yeast Interacting Proteins Database: YOR117W, YJL184W [Yeast Interacting Proteins Database

    Full Text Available c stress response, telomere uncapping and elongation, transcription; component of the EKC/KEOPS protein comp...n proposed to be involved in the modification of N-linked oligosaccharides, osmotic stress response, telomere uncap

  5. Yeast Interacting Proteins Database: YER081W, YDR105C [Yeast Interacting Proteins Database

    Full Text Available YDR105C TMS1 Vacuolar membrane protein of unknown function that is conserved in mammals; predicted to contai...tion that is conserved in mammals; predicted to contain eleven transmembrane heli

  6. Yeast Interacting Proteins Database: YKL002W, YLR423C [Yeast Interacting Proteins Database

    Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes... into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes to t

  7. Yeast Interacting Proteins Database: YKL002W, YDL165W [Yeast Interacting Proteins Database

    Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes...ins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes t

  8. Yeast Interacting Proteins Database: YLR447C, YDR277C [Yeast Interacting Proteins Database

    Full Text Available uction pathway, required for repression of transcription by Rgt1p; interacts with Rgt1p and the Snf3p and Rgt2p glucose sensors...transduction pathway, required for repression of transcription by Rgt1p; interacts with Rgt1p and the Snf3p and Rgt2p glucose sensors

  9. Yeast Interacting Proteins Database: YMR125W, YPL178W [Yeast Interacting Proteins Database

    Full Text Available so contains Sto1p, component of the spliceosomal commitment complex; interacts with Npl3p, possibly to packa...lso contains Sto1p, component of the spliceosomal commitment complex; interacts with Npl3p, possibly to pack

  10. Yeast Interacting Proteins Database: YDL239C, YGR268C [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...sembly of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spi

  11. Yeast Interacting Proteins Database: YDL239C, YPL255W [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...ediate assembly of a Don1p-containing structure at the leading edge of the prospore membrane via interaction

  12. Yeast Interacting Proteins Database: YDL239C, YOR324C [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p... a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle pole

  13. Yeast Interacting Proteins Database: YDL239C, YDR148C [Yeast Interacting Proteins Database

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...mbly of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spind

  14. THERMODYNAMICS OF PROTEIN-LIGAND INTERACTIONS AND THEIR ANALYSIS

    Rummi Devi Saini

    2017-11-01

    Full Text Available Physiological processes are controlled mainly by intermolecular recognition mechanisms which involve protein–protein and protein–ligand interactions with a high specificity and affinity to form a specific complex. Proteins being an important class of macromolecules in biological systems, it is important to understand their actions through binding to other molecules of proteins or ligands. In fact, the binding of low molecular weight ligands to proteins plays a significant role in regulating biological processes such as cellular metabolism and signal transmission. Therefore knowledge of the protein–ligand interactions and the knowledge of the mechanisms involved in the protein-ligand recognition and binding are key in understanding biology at molecular level which will facilitate the discovery, design, and development of drugs. In this review, the mechanisms involved in protein–ligand binding, the binding kinetics, thermodynamic concepts and binding driving forces are discussed. Thermodynamic mechanisms involved in a few important protein-ligand binding are described. Various spectroscopic, non-spectroscopic and computational method for analysis of protein–ligand binding are also discussed.

  15. Identification of in planta protein–protein interactions using IP-MS

    Jamge, Suraj; Angenent, Gerco; Bemer, Marian

    2018-01-01

    Gene regulation by transcription factors involves complex protein interaction networks, which include chromatin remodeling and modifying proteins as an integral part. Decoding these protein interactions is crucial for our understanding of chromatin-mediated gene regulation. Here, we describe a

  16. Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

    Hwang, Kyo Seon; Lee, Sang-Myung; Eom, Kilho; Lee, Jeong Hoon; Lee, Yoon-Sik; Park, Jung Ho; Yoon, Dae Sung; Kim, Tae Song

    2007-11-30

    We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

  17. 3DProIN: Protein-Protein Interaction Networks and Structure Visualization.

    Li, Hui; Liu, Chunmei

    2014-06-14

    3DProIN is a computational tool to visualize protein-protein interaction networks in both two dimensional (2D) and three dimensional (3D) view. It models protein-protein interactions in a graph and explores the biologically relevant features of the tertiary structures of each protein in the network. Properties such as color, shape and name of each node (protein) of the network can be edited in either 2D or 3D views. 3DProIN is implemented using 3D Java and C programming languages. The internet crawl technique is also used to parse dynamically grasped protein interactions from protein data bank (PDB). It is a java applet component that is embedded in the web page and it can be used on different platforms including Linux, Mac and Window using web browsers such as Firefox, Internet Explorer, Chrome and Safari. It also was converted into a mac app and submitted to the App store as a free app. Mac users can also download the app from our website. 3DProIN is available for academic research at http://bicompute.appspot.com.

  18. Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components.

    Timasheff, Serge N

    2002-07-23

    Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water-protein-cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein-water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration.

  19. Computational prediction of protein-protein interactions in Leishmania predicted proteomes.

    Antonio M Rezende

    Full Text Available The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks

  20. Energy landscape of all-atom protein-protein interactions revealed by multiscale enhanced sampling.

    Kei Moritsugu

    2014-10-01

    Full Text Available Protein-protein interactions are regulated by a subtle balance of complicated atomic interactions and solvation at the interface. To understand such an elusive phenomenon, it is necessary to thoroughly survey the large configurational space from the stable complex structure to the dissociated states using the all-atom model in explicit solvent and to delineate the energy landscape of protein-protein interactions. In this study, we carried out a multiscale enhanced sampling (MSES simulation of the formation of a barnase-barstar complex, which is a protein complex characterized by an extraordinary tight and fast binding, to determine the energy landscape of atomistic protein-protein interactions. The MSES adopts a multicopy and multiscale scheme to enable for the enhanced sampling of the all-atom model of large proteins including explicit solvent. During the 100-ns MSES simulation of the barnase-barstar system, we observed the association-dissociation processes of the atomistic protein complex in solution several times, which contained not only the native complex structure but also fully non-native configurations. The sampled distributions suggest that a large variety of non-native states went downhill to the stable complex structure, like a fast folding on a funnel-like potential. This funnel landscape is attributed to dominant configurations in the early stage of the association process characterized by near-native orientations, which will accelerate the native inter-molecular interactions. These configurations are guided mostly by the shape complementarity between barnase and barstar, and lead to the fast formation of the final complex structure along the downhill energy landscape.