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Sample records for identify protein partners

  1. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    Science.gov (United States)

    Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

    2008-01-01

    The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

  2. Identifying diabetes-related important protein targets with few interacting partners with the PageRank algorithm.

    Science.gov (United States)

    Grolmusz, Vince I

    2015-04-01

    Diabetes is a growing concern for the developed nations worldwide. New genomic, metagenomic and gene-technologic approaches may yield considerable results in the next several years in its early diagnosis, or in advances in therapy and management. In this work, we highlight some human proteins that may serve as new targets in the early diagnosis and therapy. With the help of a very successful mathematical tool for network analysis that formed the basis of the early successes of Google(TM), Inc., we analyse the human protein-protein interaction network gained from the IntAct database with a mathematical algorithm. The novelty of our approach is that the new protein targets suggested do not have many interacting partners (so, they are not hubs or super-hubs), so their inhibition or promotion probably will not have serious side effects. We have identified numerous possible protein targets for diabetes therapy and/or management; some of these have been well known for a long time (these validate our method), some of them appeared in the literature in the last 12 months (these show the cutting edge of the algorithm), and the remainder are still unknown to be connected with diabetes, witnessing completely new hits of the method.

  3. Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

    Science.gov (United States)

    Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

    2015-11-01

    The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

  4. Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E

    DEFF Research Database (Denmark)

    Fierro-Monti, Ivo; Mohammed, Shabaz; Matthiesen, Rune

    2006-01-01

    Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins......-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated...... levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP...

  5. Experiences of female partners of masculine-identifying trans persons.

    Science.gov (United States)

    Theron, Liesl; Collier, Kate L

    2013-01-01

    This paper explores the intimate relationship experiences of the cisgender (i.e., not transgender) female partners of masculine-identifying transgender persons, with a particular focus on these partners' self-understanding of their sexual orientation. Limited research about this topic has been conducted to date. Semi-structured interviews were conducted with eight South African women who are or have been cisgender female partners of masculine-identifying trans persons. Although the interviews showed that the relationship experiences of female partners of masculine-identifying trans persons are diverse, several common themes emerged in the narratives. The way that participants labelled their sexual orientation did not change from before to after their relationship with a transgender partner. The participants reported varied family and community responses to their relationships. Specific emotional and informational support needs for women with transgender partners were identified.

  6. PCNA Structure and Interactions with Partner Proteins

    KAUST Repository

    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.

  7. PCNA Structure and Interactions with Partner Proteins

    KAUST Repository

    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.

  8. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    Directory of Open Access Journals (Sweden)

    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

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

    International Nuclear Information System (INIS)

    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.

  10. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  11. Partner-Mediated Polymorphism of an Intrinsically Disordered Protein.

    Science.gov (United States)

    Bignon, Christophe; Troilo, Francesca; Gianni, Stefano; Longhi, Sonia

    2017-11-29

    Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (N TAIL ) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, N TAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the N TAIL /hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both N TAIL /XD and N TAIL /hsp70 interactions. Although the same N TAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased N TAIL /hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Identifying Key Attributes for Protein Beverages.

    Science.gov (United States)

    Oltman, A E; Lopetcharat, K; Bastian, E; Drake, M A

    2015-06-01

    This study identified key attributes of protein beverages and evaluated effects of priming on liking of protein beverages. An adaptive choice-based conjoint study was conducted along with Kano analysis to gain insight on protein beverage consumers (n = 432). Attributes evaluated included label claim, protein type, amount of protein, carbohydrates, sweeteners, and metabolic benefits. Utility scores for levels and importance scores for attributes were determined. Subsequently, two pairs of clear acidic whey protein beverages were manufactured that differed by age of protein source or the amount of whey protein per serving. Beverages were evaluated by 151 consumers on two occasions with or without priming statements. One priming statement declared "great flavor," the other priming statement declared 20 g protein per serving. A two way analysis of variance was applied to discern the role of each priming statement. The most important attribute for protein beverages was sweetener type, followed by amount of protein, followed by type of protein followed by label claim. Beverages with whey protein, naturally sweetened, reduced sugar and ≥15 g protein per serving were most desired. Three consumer clusters were identified, differentiated by their preferences for protein type, sweetener and amount of protein. Priming statements positively impacted concept liking (P 0.05). Consistent with trained panel profiles of increased cardboard flavor with higher protein content, consumers liked beverages with 10 g protein more than beverages with 20 g protein (6.8 compared with 5.7, P appeal. © 2015 Institute of Food Technologists®

  13. Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts.

    Directory of Open Access Journals (Sweden)

    Michael Caruso

    Full Text Available Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47 or decreased (2 association with Akt2 following insulin administration (n = 4; p<0.05. Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.

  14. Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1)

    Energy Technology Data Exchange (ETDEWEB)

    Lupo, Julien [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); CHU de Grenoble, BP217, 38043 Grenoble Cedex 9 (France); Conti, Audrey [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); Sueur, Charlotte [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); CHU de Grenoble, BP217, 38043 Grenoble Cedex 9 (France); Coly, Pierre-Alain [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); Coute, Yohann [CEA, IRTSV, Laboratoire Biologie a Grande Echelle, F-38054 Grenoble (France); INSERM, U1038, F-38054 Grenoble (France); Universite Joseph Fourier, Grenoble 1, F-38000 Grenoble Cedex 09 (France); Hunziker, Walter [Institute of Molecular and Cell Biology, Epithelial Cell Biology Laboratory, Singapore 1386473 (Singapore); Burmeister, Wim P. [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); Germi, Raphaelle [Unit of Virus Host Cell Interactions (UVHCI), UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9 (France); CHU de Grenoble, BP217, 38043 Grenoble Cedex 9 (France); Manet, Evelyne; Gruffat, Henri [INSERM U758, Unite de Virologie humaine, Lyon, 46 allee d' Italie F-69007 France (France); Ecole Normale Superieure de Lyon, F-69007 France (France); Universite Lyon1, F-69007, Lyon (France); and others

    2012-03-10

    We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

  15. Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

    Science.gov (United States)

    Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

    2013-01-01

    Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

  16. Identifying New Small Proteins in Escherichia coli.

    Science.gov (United States)

    VanOrsdel, Caitlin E; Kelly, John P; Burke, Brittany N; Lein, Christina D; Oufiero, Christopher E; Sanchez, Joseph F; Wimmers, Larry E; Hearn, David J; Abuikhdair, Fatimeh J; Barnhart, Kathryn R; Duley, Michelle L; Ernst, Sarah E G; Kenerson, Briana A; Serafin, Aubrey J; Hemm, Matthew R

    2018-04-12

    The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Towson University.

  17. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  18. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

    Science.gov (United States)

    Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I

    2010-05-01

    The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

  19. The Protein Identifier Cross-Referencing (PICR service: reconciling protein identifiers across multiple source databases

    Directory of Open Access Journals (Sweden)

    Leinonen Rasko

    2007-10-01

    Full Text Available Abstract Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR service, a web application that provides interactive and programmatic (SOAP and REST access to a mapping algorithm that uses the UniProt Archive (UniParc as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV or Microsoft Excel (XLS files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR

  20. Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle

    Science.gov (United States)

    Kaddar, Tagrid; Carreau, Madeleine

    2012-01-01

    In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these “other” nonrepair molecular functions. Thus, the goal of this paper is to revisit old ideas and to discuss protein-protein interactions related to other FA-related molecular functions to try to give the reader a wider perspective of the FA molecular puzzle. PMID:22737580

  1. Predicting protein-binding RNA nucleotides with consideration of binding partners.

    Science.gov (United States)

    Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

    2015-06-01

    In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

  2. Identifying Floppy and Rigid Regions in Proteins

    Science.gov (United States)

    Jacobs, D. J.; Thorpe, M. F.; Kuhn, L. A.

    1998-03-01

    In proteins it is possible to separate hard covalent forces involving bond lengths and bond angles from other weak forces. We model the microstructure of the protein as a generic bar-joint truss framework, where the hard covalent forces and strong hydrogen bonds are regarded as rigid bar constraints. We study the mechanical stability of proteins using FIRST (Floppy Inclusions and Rigid Substructure Topography) based on a recently developed combinatorial constraint counting algorithm (the 3D Pebble Game), which is a generalization of the 2D pebble game (D. J. Jacobs and M. F. Thorpe, ``Generic Rigidity: The Pebble Game'', Phys. Rev. Lett.) 75, 4051-4054 (1995) for the special class of bond-bending networks (D. J. Jacobs, "Generic Rigidity in Three Dimensional Bond-bending Networks", Preprint Aug (1997)). This approach is useful in identifying rigid motifs and flexible linkages in proteins, and thereby determines the essential degrees of freedom. We will show some preliminary results from the FIRST analysis on the myohemerythrin and lyozyme proteins.

  3. CDC's DELTA FOCUS Program: Identifying Promising Primary Prevention Strategies for Intimate Partner Violence.

    Science.gov (United States)

    Armstead, Theresa L; Rambo, Kirsten; Kearns, Megan; Jones, Kathryn M; Dills, Jenny; Brown, Pamela

    2017-01-01

    According to 2011 data, nearly one in four women and one in seven men in the United States experience severe physical violence by an intimate partner, creating a public health burden requiring population-level solutions. To prevent intimate partner violence (IPV) before it occurs, the CDC developed Domestic Violence Prevention Enhancements and Leadership Through Alliances, Focusing on Outcomes for Communities United with States to identify promising community- and societal-level prevention strategies to prevent IPV. The program funds 10 state domestic violence coalitions for 5 years to implement and evaluate programs and policies to prevent IPV by influencing the environments and conditions in which people live, work, and play. The program evaluation goals are to promote IPV prevention by identifying promising prevention strategies and describing those strategies using case studies, thereby creating a foundation for building practice-based evidence with a health equity approach.

  4. Identification and characterization of new molecular partners for the protein arginine methyltransferase 6 (PRMT6.

    Directory of Open Access Journals (Sweden)

    Alessandra Lo Sardo

    Full Text Available PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using invitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6.

  5. Social support needs identified by mothers affected by intimate partner violence.

    Science.gov (United States)

    Letourneau, Nicole; Morris, Catherine Young; Stewart, Miriam; Hughes, Jean; Critchley, Kim A; Secco, Loretta

    2013-09-01

    In order to offer optimal supports and services for mothers affected by intimate partner violence (IPV), an understanding of these mothers' perceptions of support needs, resources, barriers to support, and preferences for support intervention is warranted. Moreover, the growing recognition of the effects of IPV on maternal-infant relationships and of the importance of these early relationships to long-term child health outcomes suggests interventions are needed to support optimal maternal-infant relationships in these families. Thus, 64 mothers exposed to IPV when their infants were below 12 months of age participated in a retrospective qualitative study to identify mothers' support needs, resources, barriers to support, and preferences for specific support interventions to promote optimal mother-infant relationships. Participants identified both personal needs (including needs for leaving or staying with the violent partner), along with intertwined needs to care for, and help, their infants cope with the experience of violence. Mothers reported that integrated services that include information and practical support from professionals with emotional and affirmation support from peers would promote positive, nurturing mother-infant relationships and healthy child development.

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

    Science.gov (United States)

    Laine, Elodie

    2016-01-01

    ABSTRACT Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico‐chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross‐docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S‐index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface‐based (ranking) score to discriminate partners from non‐interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137–154. © 2016 Wiley Periodicals, Inc. PMID:27802579

  7. Merging in-silico and in vitro salivary protein complex partners using the STRING database: A tutorial.

    Science.gov (United States)

    Crosara, Karla Tonelli Bicalho; Moffa, Eduardo Buozi; Xiao, Yizhi; Siqueira, Walter Luiz

    2018-01-16

    Protein-protein interaction is a common physiological mechanism for protection and actions of proteins in an organism. The identification and characterization of protein-protein interactions in different organisms is necessary to better understand their physiology and to determine their efficacy. In a previous in vitro study using mass spectrometry, we identified 43 proteins that interact with histatin 1. Six previously documented interactors were confirmed and 37 novel partners were identified. In this tutorial, we aimed to demonstrate the usefulness of the STRING database for studying protein-protein interactions. We used an in-silico approach along with the STRING database (http://string-db.org/) and successfully performed a fast simulation of a novel constructed histatin 1 protein-protein network, including both the previously known and the predicted interactors, along with our newly identified interactors. Our study highlights the advantages and importance of applying bioinformatics tools to merge in-silico tactics with experimental in vitro findings for rapid advancement of our knowledge about protein-protein interactions. Our findings also indicate that bioinformatics tools such as the STRING protein network database can help predict potential interactions between proteins and thus serve as a guide for future steps in our exploration of the Human Interactome. Our study highlights the usefulness of the STRING protein database for studying protein-protein interactions. The STRING database can collect and integrate data about known and predicted protein-protein associations from many organisms, including both direct (physical) and indirect (functional) interactions, in an easy-to-use interface. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

    Science.gov (United States)

    Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

    2017-02-01

    The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

  9. Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

    Directory of Open Access Journals (Sweden)

    Inna A. Abdeeva

    2018-04-01

    Full Text Available The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57 in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1 and putative protein phosphatase (At2g30020; АТ2, whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1 and RmlC-like cupin (At5g39120. The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1 and clathrin adaptor complex-related protein (At5g05010. Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

  10. Intact Marriages in which One Partner Dis-Identifies with Experiences of Same-Sex Attraction.

    Science.gov (United States)

    Yarhouse, Mark A.; Pawlowski, Lisa M.; Tan, Erica S. N.

    2003-01-01

    This study is of heterosexually married couples in which one partner reports having experienced same-sex attraction and both partners report satisfaction with their marriage despite facing such constraints. Analysis suggested a number of themes related to how spouses learned about their partners' experiences of same-sex attraction, motivations for…

  11. Lysosomal-associated transmembrane protein 5 (LAPTM5 is a molecular partner of CD1e.

    Directory of Open Access Journals (Sweden)

    Catherine Angénieux

    Full Text Available The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

  12. Identifying intimate partner violence (IPV) during the postpartum period in a Greek sample.

    Science.gov (United States)

    Vivilaki, Victoria G; Dafermos, Vassilis; Daglas, Maria; Antoniou, Evagelia; Tsopelas, Nicholas D; Theodorakis, Pavlos N; Brown, Judith B; Lionis, Christos

    2010-12-01

    Research has highlighted the wide impact of intimate partner violence (IPV) and the public health role of community health professionals in detection of victimized women. The purpose of this study was to identify postpartum emotional and physical abuse and to validate the Greek version of the Women Abuse Screening Tool (WAST) along with its sensitivity and specificity. Five hundred seventy-nine mothers within 12 weeks postpartum were recruited from the perinatal care registers of the Maternity Departments of two public hospitals in Athens, Greece. Participants were randomly selected by clinic or shift. The WAST and the Partner Violence Screen (PVS) surveys were administered in random order to the mothers from September 2007 to January 2008. The WAST was compared with the PVS as a criterion standard. Agreement between the screening instruments was examined. The psychometric measurements that were performed included: two independent sample t tests, reliability coefficients, explanatory factor analysis using a Varimax rotation, and Principal Components Method. Confirmatory analysis-also called structural equation modeling-of principal components was conducted by Linear Structural Relations. A receiver operating characteristic (ROC) analysis was carried out to evaluate the global functioning of the scale. Two hundred four (35.6%) of the mothers screened were identified as experiencing IPV. Scores on the WAST correlated well with those on the PVS; the internal consistency of the WAST Greek version-tested using Cronbach's alpha coefficient-was found to be 0.926 and that of Guttman's split-half coefficient was 0.924. Our findings confirm the multidimensionality of the WAST, demonstrating a two-factor structure. The area under ROC curve (AUC) was found to be 0.824, and the logistic estimate for the threshold score of 0/1 fitted the model sensitivity at 99.7% and model specificity at 64.4%. Our data confirm the validity of the Greek version of the WAST in identifying IPV

  13. Identifying signs and symptoms of intimate partner violence in an oncology setting.

    Science.gov (United States)

    Mick, JoAnn

    2006-08-01

    Domestic violence (DV), or intimate partner violence (IPV), is a prevailing problem in public health. Often, healthcare providers may be the first people that victims of DV will approach to reveal their problem or seek assistance. IPV is a pattern of control using assault and intimidating behaviors that has devastating effects on individuals, their families, and communities. Oncology nurses need to become familiar with common indicators of DV so that signs and symptoms of abuse can be identified when assessing patients in an oncology setting. Standards of oncology nursing practice support that the psychosocial impact of cancer on patients and their families or significant others needs to be considered at all stages of diagnosis and treatment. The psychosocial impact of other personal situations or concerns, such as IPV, can add to the complexity of cancer management. Routine screening for signs and symptoms of psychosocial distress helps identify patients who require additional interventions. Oncology nursing practice is based on a holistic approach to patient care, which supports that identification of physical and psychosocial needs are equally important. Oncology nursing provides many unique opportunities to help patients cope with cancer. Routine nursing assessment for signs and symptoms of abuse will provide an opportunity to assist patients with cancer to manage not only the life-threatening aspects of their diagnosis but also the life-threatening aspects of IPV.

  14. Association of intimate partner violence and health-care provider-identified obesity.

    Science.gov (United States)

    Davies, Rhian; Lehman, Erik; Perry, Amanda; McCall-Hosenfeld, Jennifer S

    2016-07-01

    The association of physical and nonphysical intimate partner violence (IPV) with obesity was examined. Women (N = 1,179) were surveyed regarding demographics, obesity, and IPV exposure using humiliate-afraid-rape-kick (HARK), an IPV screening tool. A three-level lifetime IPV exposure variable measured physical, nonphysical or no IPV. Health-care provider-identified obesity was defined if participants were told by a medical provider within the past 5 years that they were obese. Bivariate analyses examined obesity by IPV and demographics. Multivariable logistic regression assessed odds of obesity by IPV type, adjusting for age, race/ethnicity, education, and marital status. Among participants, 44% reported lifetime IPV (25% physical, 19% nonphysical), and 24% reported health-care provider-identified obesity. In unadjusted analyses, obesity was more prevalent among women exposed to physical IPV (30%) and nonphysical IPV (27%), compared to women without IPV (20%, p = .002). In multivariable models, women reporting physical IPV had 1.67 times greater odds of obesity (95% confidence interval [CI] 1.20, 2.33), and women reporting nonphysical IPV had 1.46 times greater odds of obesity (95% CI 1.01, 2.10), compared to women reporting no exposure. This study extends prior data by showing, not only an association between physical IPV and obesity, but also an association between obesity and nonphysical IPV.

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

    Science.gov (United States)

    Bhardwaj, Deepak; Lakhanpaul, Suman; Tuteja, Narendra

    2012-09-01

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

  16. Testing the woman abuse screening tool to identify intimate partner violence in Indonesia.

    Science.gov (United States)

    Iskandar, Livia; Braun, Kathryn L; Katz, Alan R

    2015-04-01

    Intimate Partner Violence (IPV) is a global public health problem. IPV prevalence in Indonesia has been estimated to be less than 1%, based on reported cases. It is likely that IPV prevalence is underreported in Indonesia, as it is in many other countries. Screening for IPV has been found to increase IPV identification, but no screening tools are in use in Indonesia. The aim of this study was to test the translated Woman Abuse Screening Tool (WAST) for detecting IPV in Indonesia. The WAST was tested against a diagnostic interview by a trained psychologist on 240 women attending two Primary Health Centers in Jakarta. IPV prevalence and the reliability, sensitivity, and specificity of the WAST were estimated. Prevalence of IPV by diagnostic interview was 36.3%, much higher than published estimates. The most common forms of IPV identified were psychological (85%) and physical abuse (24%). Internal reliability of the WAST was high (α = .801). A WAST score of 13 (out of 24) is the recommended cutoff for identifying IPV, but only 17% of the Indonesian sample scored 13 or higher. Test sensitivity of the WAST with a cutoff score of 13 was only 41.9%, with a specificity of 96.8%. With a cutoff score of 10, the sensitivity improved to 84.9%, while the specificity decreased to 61.0%. Use of the WAST with a cutoff score of 10 provides good sensitivity and reasonable specificity and would provide a much-needed screening tool for use in Indonesia. Although a lower cutoff would yield a greater proportion of false positives, most of the true cases would be identified, increasing the possibility that women experiencing abuse would receive needed assistance. © The Author(s) 2014.

  17. Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

    Directory of Open Access Journals (Sweden)

    Hanzlik Robert P

    2009-06-01

    Full Text Available Abstract Background Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity. Results Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein. Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine content and protein instability index (i.e., rate of turnover in vivo were among the features most important to determining susceptibility. Conclusion As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.

  18. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    Science.gov (United States)

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  19. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    International Nuclear Information System (INIS)

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

    2007-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

  20. Interaction of Proteins Identified in Human Thyroid Cells

    Science.gov (United States)

    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

  1. Interaction of Proteins Identified in Human Thyroid Cells

    Directory of Open Access Journals (Sweden)

    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.

  2. Identifying Bacterial Immune Evasion Proteins Using Phage Display.

    Science.gov (United States)

    Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

    2017-01-01

    Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

  3. A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

    Science.gov (United States)

    Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

    2010-12-01

    Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

  4. A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

    Science.gov (United States)

    Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

    2010-01-01

    Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

  5. Bound water at protein-protein interfaces: partners, roles and hydrophobic bubbles as a conserved motif.

    Directory of Open Access Journals (Sweden)

    Mostafa H Ahmed

    Full Text Available There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region.A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules.The water molecules were found to be involved in: a (bridging interactions with both proteins (21%, b favorable interactions with only one protein (53%, and c no interactions with either protein (26%. This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins' interaction (-0.46 kcal mol(-1, but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol(-1. Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Å(2 and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or "hydrophobic bubbles". Such water molecules may have an important biological purpose in mediating protein-protein interactions.

  6. Exploiting genomic data to identify proteins involved in abalone reproduction.

    Science.gov (United States)

    Mendoza-Porras, Omar; Botwright, Natasha A; McWilliam, Sean M; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

    2014-08-28

    Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Automatically identifying gene/protein terms in MEDLINE abstracts.

    Science.gov (United States)

    Yu, Hong; Hatzivassiloglou, Vasileios; Rzhetsky, Andrey; Wilbur, W John

    2002-01-01

    Natural language processing (NLP) techniques are used to extract information automatically from computer-readable literature. In biology, the identification of terms corresponding to biological substances (e.g., genes and proteins) is a necessary step that precedes the application of other NLP systems that extract biological information (e.g., protein-protein interactions, gene regulation events, and biochemical pathways). We have developed GPmarkup (for "gene/protein-full name mark up"), a software system that automatically identifies gene/protein terms (i.e., symbols or full names) in MEDLINE abstracts. As a part of marking up process, we also generated automatically a knowledge source of paired gene/protein symbols and full names (e.g., LARD for lymphocyte associated receptor of death) from MEDLINE. We found that many of the pairs in our knowledge source do not appear in the current GenBank database. Therefore our methods may also be used for automatic lexicon generation. GPmarkup has 73% recall and 93% precision in identifying and marking up gene/protein terms in MEDLINE abstracts. A random sample of gene/protein symbols and full names and a sample set of marked up abstracts can be viewed at http://www.cpmc.columbia.edu/homepages/yuh9001/GPmarkup/. Contact. hy52@columbia.edu. Voice: 212-939-7028; fax: 212-666-0140.

  8. A partner-related risk behavior index to identify people at elevated risk for sexually transmitted infections.

    Science.gov (United States)

    Crosby, Richard; Shrier, Lydia A

    2013-04-01

    The purpose of this study was to develop and test a sexual-partner-related risk behavior index to identify high-risk individuals most likely to have a sexually transmitted infection (STI). Patients from five STI and adolescent medical clinics in three US cities were recruited (N = 928; M age = 29.2 years). Data were collected using audio-computer-assisted self-interviewing. Of seven sexual-partner-related variables, those that were significantly associated with the outcomes were combined into a partner-related risk behavior index. The dependent variables were laboratory-confirmed infection with Chlamydia trachomatis, Neisseria gonorrhoeae, and/or Trichomonas vaginalis. Nearly one-fifth of the sample (169/928; 18.4%) tested positive for an STI. Three of the seven items were significantly associated with having one or more STIs: sex with a newly released prisoner, sex with a person known or suspected of having an STI, and sexual concurrency. In combined form, this three-item index was significantly associated with STI prevalence (p one or more of three STIs. This index could be used to prioritize and guide intensified clinic-based counseling for high-risk patients of STI and other clinics.

  9. Identification of new binding partners of the chemosensory signalling protein Gγ13 expressed in taste and olfactory sensory cells.

    Directory of Open Access Journals (Sweden)

    Zhenhui eLiu

    2012-06-01

    Full Text Available Tastant detection in the oral cavity involves selective receptors localized at the apical extremity of a subset of specialized taste bud cells called taste receptor cells (TRCs. The identification of the genes coding for the taste receptors involved in this process have greatly improved our understanding of the molecular mechanisms underlying detection. However, how these receptors signal in TRCs, and whether the components of the signaling cascades interact with each other or are organized in complexes is mostly unexplored. Here we report on the identification of three new binding partners for the mouse G protein gamma 13 subunit (Gγ13, a component of the bitter taste receptors signalling cascade. For two of these Gγ13 associated proteins, namely GOPC and MPDZ, we describe the expression in taste bud cells for the first time. Furthermore, we demonstrate by means of a yeast two-hybrid interaction assay that the C terminal PDZ binding motif of Gγ13 interacts with selected PDZ domains in these proteins. In the case of the PDZ domain-containing protein zona occludens-1 (ZO-1, a major component of the tight junction defining the boundary between the apical and baso-lateral region of TRCs, we identified the first PDZ domain as the site of strong interaction with Gγ13. This association was further confirmed by co-immunoprecipitation experiments in HEK 293 cells. In addition, we present immunohistological data supporting partial co-localization of GOPC, MPDZ or ZO-1 and Gγ13 in taste buds cells. Finally, we extend this observation to olfactory sensory neurons, another type of chemosensory cells known to express both ZO-1 and Gγ13. Taken together our results implicate these new interaction partners in the sub-cellular distribution of Gγ13 in olfactory and gustatory primary sensory cells.

  10. SitesIdentify: a protein functional site prediction tool

    Directory of Open Access Journals (Sweden)

    Doig Andrew J

    2009-11-01

    Full Text Available Abstract Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify, based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/

  11. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    International Nuclear Information System (INIS)

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

    2006-01-01

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

  12. Improvements in the Protein Identifier Cross-Reference service.

    Science.gov (United States)

    Wein, Samuel P; Côté, Richard G; Dumousseau, Marine; Reisinger, Florian; Hermjakob, Henning; Vizcaíno, Juan A

    2012-07-01

    The Protein Identifier Cross-Reference (PICR) service is a tool that allows users to map protein identifiers, protein sequences and gene identifiers across over 100 different source databases. PICR takes input through an interactive website as well as Representational State Transfer (REST) and Simple Object Access Protocol (SOAP) services. It returns the results as HTML pages, XLS and CSV files. It has been in production since 2007 and has been recently enhanced to add new functionality and increase the number of databases it covers. Protein subsequences can be Basic Local Alignment Search Tool (BLAST) against the UniProt Knowledgebase (UniProtKB) to provide an entry point to the standard PICR mapping algorithm. In addition, gene identifiers from UniProtKB and Ensembl can now be submitted as input or mapped to as output from PICR. We have also implemented a 'best-guess' mapping algorithm for UniProt. In this article, we describe the usefulness of PICR, how these changes have been implemented, and the corresponding additions to the web services. Finally, we explain that the number of source databases covered by PICR has increased from the initial 73 to the current 102. New resources include several new species-specific Ensembl databases as well as the Ensembl Genome ones. PICR can be accessed at http://www.ebi.ac.uk/Tools/picr/.

  13. The reaction of neuroglobin with potential redox protein partners cytochrome b5  and cytochrome c

    DEFF Research Database (Denmark)

    Fago, Angela; Mathews, A.J.; Moens, L.

    2006-01-01

    Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b5 is relatively slow (k=6×102M-1s-1 at pH 7.0) and thus is unlikely to be of physiological...... significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2×107M-1s-1) with an apparent overall equilibrium constant of 1μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis...

  14. Probabilistic analysis for identifying the driving force of protein folding

    Science.gov (United States)

    Tokunaga, Yoshihiko; Yamamori, Yu; Matubayasi, Nobuyuki

    2018-03-01

    Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.

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

    Directory of Open Access Journals (Sweden)

    Matt eGeisler

    2015-06-01

    Full Text Available Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6,004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize.

  16. The sensitivity and specificity of four questions (HARK to identify intimate partner violence: a diagnostic accuracy study in general practice

    Directory of Open Access Journals (Sweden)

    Feder Gene

    2007-08-01

    Full Text Available Abstract Background Intimate partner violence (IPV including physical, sexual and emotional violence, causes short and long term ill-health. Brief questions that reliably identify women experiencing IPV who present in clinical settings are a pre-requisite for an appropriate response from health services to this substantial public health problem. We estimated the sensitivity and specificity of four questions (HARK developed from the Abuse Assessment screen, compared to a 30-item abuse questionnaire, the Composite Abuse Scale (CAS. Methods We administered the four HARK questions and the CAS to women approached by two researchers in general practice waiting rooms in Newham, east London. Inclusions: women aged more than 17 years waiting to see a doctor or nurse, who had been in an intimate relationship in the last year. Exclusions: women who were accompanied by children over four years of age or another adult, too unwell to complete the questionnaires, unable to understand English or unable to give informed consent. Results Two hundred and thirty two women were recruited. The response rate was 54%. The prevalence of current intimate partner violence, within the last 12 months, using the CAS cut off score of ≥3, was 23% (95% C.I. 17% to 28% with pre-test odds of 0.3 (95% C.I. 0.2 to 0.4. The receiver operator characteristic curve demonstrated that a HARK cut off score of ≥1 maximises the true positives whilst minimising the false positives. The sensitivity of the optimal HARK cut-off score of ≥1 was 81% (95% C.I. 69% to 90%, specificity 95% (95% C.I. 91% to 98%, positive predictive value 83% (95% C.I. 70% to 91%, negative predictive value 94% (95% C.I. 90% to 97%, likelihood ratio 16 (95% C.I. 8 to 31 and post-test odds 5. Conclusion The four HARK questions accurately identify women experiencing IPV in the past year and may help women disclose abuse in general practice. The HARK questions could be incorporated into the electronic medical record

  17. Ethics in Community-University-Artist Partnered Research: Tensions, Contradictions and Gaps Identified in an 'Arts for Social Change' Project.

    Science.gov (United States)

    Yassi, Annalee; Spiegel, Jennifer Beth; Lockhart, Karen; Fels, Lynn; Boydell, Katherine; Marcuse, Judith

    Academics from diverse disciplines are recognizing not only the procedural ethical issues involved in research, but also the complexity of everyday "micro" ethical issues that arise. While ethical guidelines are being developed for research in aboriginal populations and low-and-middle-income countries, multi-partnered research initiatives examining arts-based interventions to promote social change pose a unique set of ethical dilemmas not yet fully explored. Our research team, comprising health, education, and social scientists, critical theorists, artists and community-activists launched a five-year research partnership on arts-for-social change. Funded by the Social Science and Humanities Research Council in Canada and based in six universities, including over 40 community-based collaborators, and informed by five main field projects (circus with street youth, theatre by people with disabilities, dance for people with Parkinson's disease, participatory theatre with refugees and artsinfused dialogue), we set out to synthesize existing knowledge and lessons we learned. We summarized these learnings into 12 key points for reflection, grouped into three categories: community-university partnership concerns ( n  = 3), dilemmas related to the arts ( n  = 5), and team issues ( n  = 4). In addition to addressing previous concerns outlined in the literature (e.g., related to consent, anonymity, dangerous emotional terrain, etc.), we identified power dynamics (visible and hidden) hindering meaningful participation of community partners and university-based teams that need to be addressed within a reflective critical framework of ethical practice. We present how our team has been addressing these issues, as examples of how such concerns could be approached in community-university partnerships in arts for social change.

  18. Depression in Intimate Partner Violence Victims in Slovenia: A Crippling Pattern of Factors Identified in Family Practice Attendees

    Directory of Open Access Journals (Sweden)

    Nena Kopčavar Guček

    2018-01-01

    Full Text Available This multi-centre cross-sectional study explored associations between prevalence of depression and exposure to intimate partner violence (IPV at any time in patients’ adult life in 471 participants of a previous IPV study. In 2016, 174 interviews were performed, using the Short Form Domestic Violence Exposure Questionnaire, the Zung Scale and questions about behavioural patterns of exposure to IPV. Family doctors reviewed patients’ medical charts for period from 2012 to 2016, using the Domestic Violence Exposure Medical Chart Check List, for conditions which persisted for at least three years. Depression was found to be associated with any exposure to IPV in adult life and was more likely to affect women. In multivariable logistic regression modelling, factors associated with self-rated depression were identified (p < 0.05. Exposure to emotional and physical violence was identified as a risk factor in the first model, explaining 23% of the variance. The second model explained 66% of the variance; past divorce, dysfunctional family relationships and a history of incapacity to work increased the likelihood of depression in patients. Family doctors should consider IPV exposure when detecting depression, since lifetime IPV exposure was found to be 40.4% and 36.9% of depressed revealed it.

  19. Depression in Intimate Partner Violence Victims in Slovenia: A Crippling Pattern of Factors Identified in Family Practice Attendees.

    Science.gov (United States)

    Guček, Nena Kopčavar; Selič, Polona

    2018-01-26

    This multi-centre cross-sectional study explored associations between prevalence of depression and exposure to intimate partner violence (IPV) at any time in patients' adult life in 471 participants of a previous IPV study. In 2016, 174 interviews were performed, using the Short Form Domestic Violence Exposure Questionnaire, the Zung Scale and questions about behavioural patterns of exposure to IPV. Family doctors reviewed patients' medical charts for period from 2012 to 2016, using the Domestic Violence Exposure Medical Chart Check List, for conditions which persisted for at least three years. Depression was found to be associated with any exposure to IPV in adult life and was more likely to affect women. In multivariable logistic regression modelling, factors associated with self-rated depression were identified (p < 0.05). Exposure to emotional and physical violence was identified as a risk factor in the first model, explaining 23% of the variance. The second model explained 66% of the variance; past divorce, dysfunctional family relationships and a history of incapacity to work increased the likelihood of depression in patients. Family doctors should consider IPV exposure when detecting depression, since lifetime IPV exposure was found to be 40.4% and 36.9% of depressed revealed it.

  20. Use of the Operon Structure of the C. elegans Genome as a Tool to Identify Functionally Related Proteins

    Directory of Open Access Journals (Sweden)

    Silvia Dossena

    2013-12-01

    Full Text Available One of the most pressing challenges in the post genomic era is the identification and characterization of protein-protein interactions (PPIs, as these are essential in understanding the cellular physiology of health and disease. Experimental techniques suitable for characterizing PPIs (X-ray crystallography or nuclear magnetic resonance spectroscopy, among others are usually laborious, time-consuming and often difficult to apply to membrane proteins, and therefore require accurate prediction of the candidate interacting partners. High-throughput experimental methods (yeast two-hybrid and affinity purification succumb to the same shortcomings, and can also lead to high rates of false positive and negative results. Therefore, reliable tools for predicting PPIs are needed. The use of the operon structure in the eukaryote Caenorhabditis elegans genome is a valuable, though underserved, tool for identifying physically or functionally interacting proteins. Based on the concept that genes organized in the same operon may encode physically or functionally related proteins, this algorithm is easy to be applied and, importantly, gives a limited number of candidate partners of a given protein, allowing for focused experimental verification. Moreover, this approach can be successfully used to predict PPIs in the human system, including those of membrane proteins.

  1. A coevolution analysis for identifying protein-protein interactions by Fourier transform

    Science.gov (United States)

    Yin, Changchuan; Yau, Stephen S. -T.

    2017-01-01

    Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

  2. A coevolution analysis for identifying protein-protein interactions by Fourier transform.

    Directory of Open Access Journals (Sweden)

    Changchuan Yin

    Full Text Available Protein-protein interactions (PPIs play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA; however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40. The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI.

  3. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    Directory of Open Access Journals (Sweden)

    Jun Ren

    2014-01-01

    Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

  4. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  5. A lanthipeptide library used to identify a protein-protein interaction inhibitor.

    Science.gov (United States)

    Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

    2018-04-01

    In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

  6. Identify drug repurposing candidates by mining the protein data bank.

    Science.gov (United States)

    Moriaud, Fabrice; Richard, Stéphane B; Adcock, Stewart A; Chanas-Martin, Laetitia; Surgand, Jean-Sébastien; Ben Jelloul, Marouane; Delfaud, François

    2011-07-01

    Predicting off-targets by computational methods is gaining increasing interest in early-stage drug discovery. Here, we present a computational method based on full 3D comparisons of 3D structures. When a similar binding site is detected in the Protein Data Bank (PDB) (or any protein structure database), it is possible that the corresponding ligand also binds to that similar site. On one hand, this target hopping case is probably rare because it requires a high similarity between the binding sites. On the other hand, it could be a strong rational evidence to highlight possible off-target reactions and possibly a potential undesired side effect. This target-based drug repurposing can be extended a significant step further with the capability of searching the full surface of all proteins in the PDB, and therefore not relying on pocket detection. Using this approach, we describe how MED-SuMo reproduces the repurposing of tadalafil from PDE5A to PDE4A and a structure of PDE4A with tadalafil. Searching for local protein similarities generates more hits than for whole binding site similarities and therefore fragment repurposing is more likely to occur than for drug-sized compounds. In this work, we illustrate that by mining the PDB for proteins sharing similarities with the hinge region of protein kinases. The experimentally validated examples, biotin carboxylase and synapsin, are retrieved. Further to fragment repurposing, this approach can be applied to the detection of druggable sites from 3D structures. This is illustrated with detection of the protein kinase hinge motif in the HIV-RT non-nucleosidic allosteric site.

  7. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    Science.gov (United States)

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  8. Identifying Novel Candidate Genes Related to Apoptosis from a Protein-Protein Interaction Network

    Directory of Open Access Journals (Sweden)

    Baoman Wang

    2015-01-01

    Full Text Available Apoptosis is the process of programmed cell death (PCD that occurs in multicellular organisms. This process of normal cell death is required to maintain the balance of homeostasis. In addition, some diseases, such as obesity, cancer, and neurodegenerative diseases, can be cured through apoptosis, which produces few side effects. An effective comprehension of the mechanisms underlying apoptosis will be helpful to prevent and treat some diseases. The identification of genes related to apoptosis is essential to uncover its underlying mechanisms. In this study, a computational method was proposed to identify novel candidate genes related to apoptosis. First, protein-protein interaction information was used to construct a weighted graph. Second, a shortest path algorithm was applied to the graph to search for new candidate genes. Finally, the obtained genes were filtered by a permutation test. As a result, 26 genes were obtained, and we discuss their likelihood of being novel apoptosis-related genes by collecting evidence from published literature.

  9. Identifying and Intervening with Substance-Using Women Exposed to Intimate Partner Violence: Phenomenology, Comorbidities, and Integrated Approaches Within Primary Care and Other Agency Settings

    OpenAIRE

    Weaver, Terri L.; Gilbert, Louisa; El-Bassel, Nabila; Resnick, Heidi S.; Noursi, Samia

    2015-01-01

    Substance use and/or disorders (SUDs) have been identified as a significant correlate of intimate partner violence (IPV) exposure and present complex issues that intersect with the topography of IPV, attendant mental health, and physical co-morbidities and may pose barriers to primary care- and other agency-based screening and intervention efforts. Despite substantial research indicating significantly higher rates of all types and severity of IPV victimization among women with SUDs and bidire...

  10. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

    Directory of Open Access Journals (Sweden)

    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  11. Target and identify: triazene linker helps identify azidation sites of labelled proteins via click and cleave strategy.

    Science.gov (United States)

    Lohse, Jonas; Schindl, Alexandra; Danda, Natasha; Williams, Chris P; Kramer, Karl; Kuster, Bernhard; Witte, Martin D; Médard, Guillaume

    2017-10-31

    A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA.

  12. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

  13. The presence of the sexual partner and nutritional condition alter the Anastrepha obliqua MacQuart (Diptera: Tephritidae) protein discrimination threshold

    Energy Technology Data Exchange (ETDEWEB)

    Cresoni-Pereira, Carla; Zucoloto, Fernando S. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Filosofia, Ciencias e Letras. Dept. de Biologia

    2005-11-15

    The minimum protein amount that Anastrepha obliqua MacQuart can detect in its alimentary source is variable, though the causes of such variation are not very well known. In this study, the authors tested whether the sexual partners nutritional condition and presence devoid of direct contact alter the A. obliqua protein discrimination threshold. Male and female insects were assigned to groups as follows: (1) newly emerged, (2) deprived of protein source (yeast) during 18 days, (3) non-yeast-deprived during 18 days, (4) yeast-deprived in the presence of equally yeast-deprived sexual partners, (5) yeast-deprived in the presence of non-yeast-deprived partners, (6) non-yeast-deprived with yeast-deprived partners and (7) non-yeast-deprived with non-yeast-deprived partners. The sexual partners were maintained apart by a transparent plastic screen with small holes. Not only the males presence but also their nutritional condition have altered the females discrimination threshold, particularly when the females were deprived and when non- deprived females cohabited with deprived males. Therefore, the females threshold was determined by their own nutritional condition in addition to recognition of the males nutritional condition. The males discrimination threshold was higher for non-deprived subjects than for the deprived ones. The occurrence of responses in the absence of direct contact between males and females has shown that they may use a chemical mechanism for mutual recognition of the sexual partner nutritional condition. (author)

  14. The presence of the sexual partner and nutritional condition alter the Anastrepha obliqua MacQuart (Diptera: Tephritidae) protein discrimination threshold

    International Nuclear Information System (INIS)

    Cresoni-Pereira, Carla; Zucoloto, Fernando S.

    2005-01-01

    The minimum protein amount that Anastrepha obliqua MacQuart can detect in its alimentary source is variable, though the causes of such variation are not very well known. In this study, the authors tested whether the sexual partners nutritional condition and presence devoid of direct contact alter the A. obliqua protein discrimination threshold. Male and female insects were assigned to groups as follows: (1) newly emerged, (2) deprived of protein source (yeast) during 18 days, (3) non-yeast-deprived during 18 days, (4) yeast-deprived in the presence of equally yeast-deprived sexual partners, (5) yeast-deprived in the presence of non-yeast-deprived partners, (6) non-yeast-deprived with yeast-deprived partners and (7) non-yeast-deprived with non-yeast-deprived partners. The sexual partners were maintained apart by a transparent plastic screen with small holes. Not only the males presence but also their nutritional condition have altered the females discrimination threshold, particularly when the females were deprived and when non- deprived females cohabited with deprived males. Therefore, the females threshold was determined by their own nutritional condition in addition to recognition of the males nutritional condition. The males discrimination threshold was higher for non-deprived subjects than for the deprived ones. The occurrence of responses in the absence of direct contact between males and females has shown that they may use a chemical mechanism for mutual recognition of the sexual partner nutritional condition. (author)

  15. HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

    Directory of Open Access Journals (Sweden)

    Hegemann Johannes H

    2005-05-01

    Full Text Available Background HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. Results An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the

  16. Identifying and intervening with substance-using women exposed to intimate partner violence: phenomenology, comorbidities, and integrated approaches within primary care and other agency settings.

    Science.gov (United States)

    Weaver, Terri L; Gilbert, Louisa; El-Bassel, Nabila; Resnick, Heidi S; Noursi, Samia

    2015-01-01

    Substance use and/or disorders (SUDs) have been identified as a significant correlate of intimate partner violence (IPV) exposure and present complex issues that intersect with the topography of IPV, attendant mental health, and physical co-morbidities and may pose barriers to primary care- and other agency-based screening and intervention efforts. Despite substantial research indicating significantly higher rates of all types and severity of IPV victimization among women with SUDs and bidirectional associations between partner or self-use of drugs or alcohol and IPV victimization, effective screening, brief interventions, coordinated systems of care, and treatment approaches to address these co-occurring problems remain very limited. We integrated select research examining the intersection of IPV victimization and SUDs and several comorbidities that have significant public health impact and provided recommendations for scaling up targeted interventions to redress these co-occurring problems among women in primary, emergency, and other care settings.

  17. Self-Presentation, Desired Partner Characteristics, and Sexual Behavior Preferences in Online Personal Advertisements of Men Seeking Non-Gay-Identified Men

    Science.gov (United States)

    Schrimshaw, Eric W.

    2015-01-01

    Despite attention to the sexual behaviors of non-gay-identified (NGI) men who have same-sex encounters, virtually no research has focused on issues of partner desirability and selection. Limited evidence suggests that a subgroup of men who have sex with men (MSM) advertise online for sexual encounters with NGI men. Exchange theory provided a framework to investigate this seeking of NGI men, based on the content of Internet personal advertisements for same-sex encounters. Researchers analyzed 282 ads posted to an online bulletin board. Ads by men who explicitly desired encounters with NGI men were compared with those by men who did not indicate this preference in potential partners. Multivariate analyses revealed that NGI-seeking men had significantly increased odds of identifying as discreet (Adjusted odds ratio [AOR] = 2.82), seeking a discreet encounter (AOR = 4.68), seeking a masculine partner (AOR = 2.18), being willing to host (AOR = 2.77), as well as seeking oral-receptive sex (AOR = 2.69), unprotected oral sex (AOR = 6.76), and anal-receptive sex (AOR = 2.18). Further, NGI-seeking ads were more likely to not mention condom use or safer sex practices (AOR = 4.13) and were less likely to indicate a desire for oral-insertive sex (AOR = 0.34) and rimming (AOR = 0.21). Findings suggest that some men may deliberately present themselves in ways that they perceive as being attractive to NGI men, and have research implications for NGI MSM, their partners, and the risk outcomes of these online ads. PMID:25750927

  18. Self-Presentation, Desired Partner Characteristics, and Sexual Behavior Preferences in Online Personal Advertisements of Men Seeking Non-Gay-Identified Men.

    Science.gov (United States)

    Downing, Martin J; Schrimshaw, Eric W

    2014-03-14

    Despite attention to the sexual behaviors of non-gay-identified (NGI) men who have same-sex encounters, virtually no research has focused on issues of partner desirability and selection. Limited evidence suggests that a subgroup of men who have sex with men (MSM) advertise online for sexual encounters with NGI men. Exchange theory provided a framework to investigate this seeking of NGI men, based on the content of Internet personal advertisements for same-sex encounters. Researchers analyzed 282 ads posted to an online bulletin board. Ads by men who explicitly desired encounters with NGI men were compared with those by men who did not indicate this preference in potential partners. Multivariate analyses revealed that NGI-seeking men had significantly increased odds of identifying as discreet (Adjusted odds ratio [ AOR ] = 2.82), seeking a discreet encounter ( AOR = 4.68), seeking a masculine partner ( AOR = 2.18), being willing to host ( AOR = 2.77), as well as seeking oral-receptive sex ( AOR = 2.69), unprotected oral sex ( AOR = 6.76), and anal-receptive sex ( AOR = 2.18). Further, NGI-seeking ads were more likely to not mention condom use or safer sex practices ( AOR = 4.13) and were less likely to indicate a desire for oral-insertive sex ( AOR = 0.34) and rimming ( AOR = 0.21). Findings suggest that some men may deliberately present themselves in ways that they perceive as being attractive to NGI men, and have research implications for NGI MSM, their partners, and the risk outcomes of these online ads.

  19. Physical interaction between the strawberry allergen Fra a 1 and an associated partner FaAP: Interaction of Fra a 1 proteins and FaAP.

    Science.gov (United States)

    Franz-Oberdorf, Katrin; Langer, Andreas; Strasser, Ralf; Isono, Erika; Ranftl, Quirin L; Wunschel, Christian; Schwab, Wilfried

    2017-10-01

    The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis-related 10 (PR-10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1-Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1-Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C-terminal sequence of FaAP showed strong auto-activation activity in yeast 2-hybrid analysis a novel time resolved DNA-switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with K D of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR-10 proteins might be integrated into a protein-interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins. © 2017 Wiley Periodicals, Inc.

  20. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  1. Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

    Science.gov (United States)

    Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

    2018-01-16

    "A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

  2. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Science.gov (United States)

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  3. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  4. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  5. Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain.

    Directory of Open Access Journals (Sweden)

    Tingjun Hou

    2006-01-01

    Full Text Available Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3 domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.

  6. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

    Science.gov (United States)

    Nixon, Ralph A

    2017-07-01

    Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

  7. Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

    Science.gov (United States)

    Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

    2013-06-01

    The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

  8. Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

    OpenAIRE

    Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

    2013-01-01

    The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present ...

  9. High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Aida Barreiro-Alonso

    2016-01-01

    Full Text Available Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised.

  10. Partner, Kristal and Dukat: A new generation of the Novi Sad spring protein pea (Pisum sativum cultivars

    Directory of Open Access Journals (Sweden)

    Mihailović Vojislav

    2009-01-01

    Full Text Available In 2007 and 2008, the trials of the Department of Variety Protection and Registration of the Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia were carried out on four locations, including three new Novi Sad spring pea line, L-536, L-537 and L-538, and the control cultivar Javor. In average, the grain yield of all three lines was higher in comparison to the control cultivar, with the highest average yield in Partner (2732 kg ha-1. All three cultivars have shown that in favourable years may give grain yields higher than 3500 kg ha-1. The crude protein content ranged from 278.3 g kg-1, in Javor, to 307.0 g kg-1, in Dukat. .

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

    Science.gov (United States)

    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.

  12. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  13. Reactions to Participating in Intimate Partner Violence and Minority Stress Research: A Mixed Methodological Study of Self-Identified Lesbian and Gay Emerging Adults.

    Science.gov (United States)

    Edwards, Katie M; Sylaska, Kateryna M

    2016-01-01

    The purpose of this study was to examine lesbian and gay (LG) young adults' reactions to participating in intimate partner violence (IPV) and minority stress research using a mixed methodological design. Participants were 277 U.S. college students currently involved in same-sex relationships and self-identified cisgender LG who completed an online questionnaire that included closed- and open-ended questions. Results suggested that IPV research was well tolerated by the vast majority of participants; close to one in 10 participants reported being upset by the study questions, yet 75% of upset individuals reported some level of personal benefit. Reasons for upset as identified in the open-ended responses included thinking about personal experiences with IPV, as the perpetrator or friend of a victim, as well as thinking about the uncertainty of their future with their current partner. The correlates of emotional reactions and personal benefits to research participation were also examined, and these varied among gay men and lesbian women. Implications of these findings underscore the importance of accurate reflection of risk and benefits in informed consent documents as well as systematic evaluation of sexual minority participants' reactions to research participation in an effort to conduct ethically sound sexual science research.

  14. Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway

    DEFF Research Database (Denmark)

    Madsen, Kenneth Lindegaard; Thorsen, Thor Seneca; Rahbek-Clemmensen, Troels

    2012-01-01

    The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing...... functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which...

  15. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    Science.gov (United States)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  16. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  17. Gene Unprediction with Spurio: A tool to identify spurious protein sequences.

    Science.gov (United States)

    Höps, Wolfram; Jeffryes, Matt; Bateman, Alex

    2018-01-01

    We now have access to the sequences of tens of millions of proteins. These protein sequences are essential for modern molecular biology and computational biology. The vast majority of protein sequences are derived from gene prediction tools and have no experimental supporting evidence for their translation.  Despite the increasing accuracy of gene prediction tools there likely exists a large number of spurious protein predictions in the sequence databases.  We have developed the Spurio tool to help identify spurious protein predictions in prokaryotes.  Spurio searches the query protein sequence against a prokaryotic nucleotide database using tblastn and identifies homologous sequences. The tblastn matches are used to score the query sequence's likelihood of being a spurious protein prediction using a Gaussian process model. The most informative feature is the appearance of stop codons within the presumed translation of homologous DNA sequences. Benchmarking shows that the Spurio tool is able to distinguish spurious from true proteins. However, transposon proteins are prone to be predicted as spurious because of the frequency of degraded homologs found in the DNA sequence databases. Our initial experiments suggest that less than 1% of the proteins in the UniProtKB sequence database are likely to be spurious and that Spurio is able to identify over 60 times more spurious proteins than the AntiFam resource. The Spurio software and source code is available under an MIT license at the following URL: https://bitbucket.org/bateman-group/spurio.

  18. Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

    Science.gov (United States)

    Bonnavion, Rémy; Teinturier, Romain; Gherardi, Samuele; Leteurtre, Emmanuelle; Yu, Run; Cordier-Bussat, Martine; Du, Rui; Pattou, François; Vantyghem, Marie-Christine; Bertolino, Philippe; Lu, Jieli; Zhang, Chang Xian

    2017-05-01

    Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

    Science.gov (United States)

    Estrada-Gómez, Sebastian; Vargas-Muñoz, Leidy Johana; Saldarriaga-Córdoba, Mónica; Cifuentes, Yeimy; Perafan, Carlos

    2017-04-01

    Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    LENUS (Irish Health Repository)

    Toomey, David

    2009-01-01

    BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins\\/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and\\/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY\\/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS\\/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under \\'change-of-application\\' patents.

  1. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    Directory of Open Access Journals (Sweden)

    David Toomey

    Full Text Available BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i homologous to previously crystallized proteins or (ii targets of known drugs, but are (iii not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under 'change-of-application' patents.

  2. Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

    International Nuclear Information System (INIS)

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-01

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  3. Semantic integration to identify overlapping functional modules in protein interaction networks

    Directory of Open Access Journals (Sweden)

    Ramanathan Murali

    2007-07-01

    Full Text Available Abstract Background The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms. Results We have developed novel metrics, called semantic similarity and semantic interactivity, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. We presented a flow-based modularization algorithm to efficiently identify overlapping modules in the weighted interaction networks. The experimental results show that the semantic similarity and semantic interactivity of interacting pairs were positively correlated with functional co-occurrence. The effectiveness of the algorithm for identifying modules was evaluated using functional categories from the MIPS database. We demonstrated that our algorithm had higher accuracy compared to other competing approaches. Conclusion The integration of protein interaction networks with GO annotation data and the capability of detecting overlapping modules substantially improve the accuracy of module identification.

  4. Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

    2017-05-01

    The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.

    Science.gov (United States)

    Lin, Jiangguo; Countryman, Preston; Buncher, Noah; Kaur, Parminder; E, Longjiang; Zhang, Yiyun; Gibson, Greg; You, Changjiang; Watkins, Simon C; Piehler, Jacob; Opresko, Patricia L; Kad, Neil M; Wang, Hong

    2014-02-01

    Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

  6. Role of OCT-1 and partner proteins in T cell differentiation.

    Science.gov (United States)

    Hwang, Soo Seok; Kim, Lark Kyun; Lee, Gap Ryol; Flavell, Richard A

    2016-06-01

    The understanding of CD4 T cell differentiation gives important insights into the control of immune responses against various pathogens and in autoimmune diseases. Naïve CD4 T cells become effector T cells in response to antigen stimulation in combination with various environmental cytokine stimuli. Several transcription factors and cis-regulatory regions have been identified to regulate epigenetic processes on chromatin, to allow the production of proper effector cytokines during CD4 T cell differentiation. OCT-1 (Pou2f1) is well known as a widely expressed transcription factor in most tissues and cells. Although the importance of OCT-1 has been emphasized during development and differentiation, its detailed molecular underpinning and precise role are poorly understood. Recently, a series of studies have reported that OCT-1 plays a critical role in CD4 T cells through regulating gene expression during differentiation and mediating long-range chromosomal interactions. In this review, we will describe the role of OCT-1 in CD4 T cell differentiation and discuss how this factor orchestrates the fate and function of CD4 effector T cells. Copyright © 2016. Published by Elsevier B.V.

  7. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

    Science.gov (United States)

    Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Method for early detection of infectious mononucleosis by identifying Inmono proteins

    Science.gov (United States)

    Willard, Karen E.

    1984-01-01

    Early detection of infectious mononucleosis is carried out using a sample of human blood by isolating and identifying the presence of Inmono proteins in the sample from a two-dimensional protein map with the proteins being characterized by having isoelectric banding as measured in urea of about -16 to -17 with respect to certain isoelectric point standards and molecular mass of about 70 to 75 K daltons as measured in the presence of sodium dodecylsulfate containing polyacrylamide gels, the presence of the Inmono proteins being correlated with the existence of infectious mononucleosis.

  9. Identifying essential proteins based on sub-network partition and prioritization by integrating subcellular localization information.

    Science.gov (United States)

    Li, Min; Li, Wenkai; Wu, Fang-Xiang; Pan, Yi; Wang, Jianxin

    2018-06-14

    Essential proteins are important participants in various life activities and play a vital role in the survival and reproduction of living organisms. Identification of essential proteins from protein-protein interaction (PPI) networks has great significance to facilitate the study of human complex diseases, the design of drugs and the development of bioinformatics and computational science. Studies have shown that highly connected proteins in a PPI network tend to be essential. A series of computational methods have been proposed to identify essential proteins by analyzing topological structures of PPI networks. However, the high noise in the PPI data can degrade the accuracy of essential protein prediction. Moreover, proteins must be located in the appropriate subcellular localization to perform their functions, and only when the proteins are located in the same subcellular localization, it is possible that they can interact with each other. In this paper, we propose a new network-based essential protein discovery method based on sub-network partition and prioritization by integrating subcellular localization information, named SPP. The proposed method SPP was tested on two different yeast PPI networks obtained from DIP database and BioGRID database. The experimental results show that SPP can effectively reduce the effect of false positives in PPI networks and predict essential proteins more accurately compared with other existing computational methods DC, BC, CC, SC, EC, IC, NC. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    Science.gov (United States)

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The flexible C-terminal arm of the Lassa arenavirus Z-protein mediates interactions with multiple binding partners.

    Science.gov (United States)

    May, Eric R; Armen, Roger S; Mannan, Aristotle M; Brooks, Charles L

    2010-08-01

    The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population-weighted ensembles of low-energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was identified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein-binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein. (c) 2010 Wiley-Liss, Inc.

  12. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    International Nuclear Information System (INIS)

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ( 3 H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of 3 H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked 3 H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked 3 H-EFDA in toluene alone, and of the protein-linked 3 H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III

  13. Identifying secondary structures in proteins using NMR chemical shift 3D correlation maps

    Science.gov (United States)

    Kumari, Amrita; Dorai, Kavita

    2013-06-01

    NMR chemical shifts are accurate indicators of molecular environment and have been extensively used as aids in protein structure determination. This work focuses on creating empirical 3D correlation maps of backbone chemical shift nuclei for use as identifiers of secondary structure elements in proteins. A correlated database of backbone nuclei chemical shifts was constructed from experimental structural data gathered from entries in the Protein Data Bank (PDB) as well as isotropic chemical shift values from the RefDB database. Rigorous statistical analysis of the maps led to the conclusion that specific correlations between triplets of backbone chemical shifts are best able to differentiate between different secondary structures such as α-helices, β-strands and turns. The method is compared with similar techniques that use NMR chemical shift information as aids in biomolecular structure determination and performs well in tests done on experimental data determined for different types of proteins, including large multi-domain proteins and membrane proteins.

  14. Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1--old partners, new players.

    Directory of Open Access Journals (Sweden)

    Evandro F Tirapelle

    Full Text Available Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB, an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1 coating the PHB granule as well as the PHB synthase (PhbC1 responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2. Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.

  15. Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

    Science.gov (United States)

    Tirapelle, Evandro F.; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z.; Kadowaki, Marco A. S.; Steffens, Maria B. R.; Monteiro, Rose A.; Souza, Emanuel M.; Pedrosa, Fabio O.; Chubatsu, Leda S.

    2013-01-01

    Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae. PMID:24086439

  16. Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1--old partners, new players.

    Science.gov (United States)

    Tirapelle, Evandro F; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z; Kadowaki, Marco A S; Steffens, Maria B R; Monteiro, Rose A; Souza, Emanuel M; Pedrosa, Fabio O; Chubatsu, Leda S

    2013-01-01

    Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.

  17. Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.

    LENUS (Irish Health Repository)

    O'Connor, Roisin

    2010-01-01

    Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

  18. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

    Directory of Open Access Journals (Sweden)

    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  19. msiDBN: A Method of Identifying Critical Proteins in Dynamic PPI Networks

    Directory of Open Access Journals (Sweden)

    Yuan Zhang

    2014-01-01

    Full Text Available Dynamics of protein-protein interactions (PPIs reveals the recondite principles of biological processes inside a cell. Shown in a wealth of study, just a small group of proteins, rather than the majority, play more essential roles at crucial points of biological processes. This present work focuses on identifying these critical proteins exhibiting dramatic structural changes in dynamic PPI networks. First, a comprehensive way of modeling the dynamic PPIs is presented which simultaneously analyzes the activity of proteins and assembles the dynamic coregulation correlation between proteins at each time point. Second, a novel method is proposed, named msiDBN, which models a common representation of multiple PPI networks using a deep belief network framework and analyzes the reconstruction errors and the variabilities across the time courses in the biological process. Experiments were implemented on data of yeast cell cycles. We evaluated our network construction method by comparing the functional representations of the derived networks with two other traditional construction methods. The ranking results of critical proteins in msiDBN were compared with the results from the baseline methods. The results of comparison showed that msiDBN had better reconstruction rate and identified more proteins of critical value to yeast cell cycle process.

  20. Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

    DEFF Research Database (Denmark)

    Sørensen, Rikke Kruse; Krantz, James; Barker, Natalie

    2017-01-01

    . The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1......, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein....... These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology....

  1. A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

    Science.gov (United States)

    Nicholson, Judith; Scherl, Alex; Way, Luke; Blackburn, Elizabeth A; Walkinshaw, Malcolm D; Ball, Kathryn L; Hupp, Ted R

    2014-06-01

    Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat

    DEFF Research Database (Denmark)

    Yang, Fen; Jensen, Jens D.; Svensson, Birte

    2012-01-01

    Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley...... or wheat flour as the sole nutrient source to mimic the host–pathogen interaction. A gel‐based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty‐nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation...... between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase‐polymerase chain reaction in barley...

  3. The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

    Directory of Open Access Journals (Sweden)

    Marina Mihaela

    2012-07-01

    Full Text Available Abstract MEK Partner 1 (MP1 or MAPKSP1 is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1’s functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

  4. Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

    Directory of Open Access Journals (Sweden)

    Trimpalis Philip

    2011-07-01

    Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

  5. Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

    Science.gov (United States)

    Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

    2014-01-01

    Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    Science.gov (United States)

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  7. Children exposed to intimate partner violence: Identifying differential effects of family environment on children's trauma and psychopathology symptoms through regression mixture models.

    Science.gov (United States)

    McDonald, Shelby Elaine; Shin, Sunny; Corona, Rosalie; Maternick, Anna; Graham-Bermann, Sandra A; Ascione, Frank R; Herbert Williams, James

    2016-08-01

    The majority of analytic approaches aimed at understanding the influence of environmental context on children's socioemotional adjustment assume comparable effects of contextual risk and protective factors for all children. Using self-reported data from 289 maternal caregiver-child dyads, we examined the degree to which there are differential effects of severity of intimate partner violence (IPV) exposure, yearly household income, and number of children in the family on posttraumatic stress symptoms (PTS) and psychopathology symptoms (i.e., internalizing and externalizing problems) among school-age children between the ages of 7-12 years. A regression mixture model identified three latent classes that were primarily distinguished by differential effects of IPV exposure severity on PTS and psychopathology symptoms: (1) asymptomatic with low sensitivity to environmental factors (66% of children), (2) maladjusted with moderate sensitivity (24%), and (3) highly maladjusted with high sensitivity (10%). Children with mothers who had higher levels of education were more likely to be in the maladjusted with moderate sensitivity group than the asymptomatic with low sensitivity group. Latino children were less likely to be in both maladjusted groups compared to the asymptomatic group. Overall, the findings suggest differential effects of family environmental factors on PTS and psychopathology symptoms among children exposed to IPV. Implications for research and practice are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. A cross-sectional study identifying the pattern of factors related to psychological intimate partner violence exposure in Slovenian family practice attendees: what hurt them the most

    Science.gov (United States)

    2014-01-01

    Background Intimate partner violence (IPV) is yet to be fully acknowledged as a public health problem in Slovenia. This study aimed to explore the health and other patient characteristics associated with psychological IPV exposure and gender-related specificity in family clinic attendees. Methods In a multi-centre cross-sectional study, 960 family practice attendees aged 18 years and above were recruited. In 689 interviews with currently- or previously-partnered patients, the short form of A Domestic Violence Exposure Questionnaire and additional questions about behavioural patterns of exposure to psychological abuse in the past year were given. General practitioners (GPs) reviewed the medical charts of 470 patients who met the IPV exposure criteria. The Domestic Violence Exposure Medical Chart Check List was used, collecting data on the patients’ lives and physical, sexual and reproductive, and psychological health status, as well as sick leave, hospitalisation, visits to family practices and referrals to other clinical specialists in the past year. In multivariate logistic regression modelling the factors associated with past year psychological IPV exposure were identified, with P < 0.05 set as the level of statistical significance. Results Of the participants (n = 470), 12.1% (n = 57) were exposed to psychological IPV in the previous year (46 women and 11 men). They expressed more complaints regarding sexual and reproductive (p = 0.011), and psychological and behavioural status (p <0.001), in the year prior to the survey. Unemployment or working part-time, a college degree, an intimate relationship of six years or more and a history of disputes in the intimate relationship, increased the odds of psychological IPV exposure in the sample, explaining 41% of the variance. In females, unemployment and a history of disputes in the intimate relationship explained 43% of the variance. Conclusions The prevalence of psychological IPV above 10% during the past year

  9. The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

    Science.gov (United States)

    Miura, Toyokazu; Takeo, Satoru; Ntege, Edward H; Otsuki, Hitoshi; Sawasaki, Tatsuya; Ishino, Tomoko; Takashima, Eizo; Tsuboi, Takafumi

    2018-06-02

    Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Mass Spectrometry-Based Methods for Identifying Oxidized Proteins in Disease: Advances and Challenges

    Directory of Open Access Journals (Sweden)

    Ivan Verrastro

    2015-04-01

    Full Text Available Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

  11. A feedback framework for protein inference with peptides identified from tandem mass spectra

    Directory of Open Access Journals (Sweden)

    Shi Jinhong

    2012-11-01

    Full Text Available Abstract Background Protein inference is an important computational step in proteomics. There exists a natural nest relationship between protein inference and peptide identification, but these two steps are usually performed separately in existing methods. We believe that both peptide identification and protein inference can be improved by exploring such nest relationship. Results In this study, a feedback framework is proposed to process peptide identification reports from search engines, and an iterative method is implemented to exemplify the processing of Sequest peptide identification reports according to the framework. The iterative method is verified on two datasets with known validity of proteins and peptides, and compared with ProteinProphet and PeptideProphet. The results have shown that not only can the iterative method infer more true positive and less false positive proteins than ProteinProphet, but also identify more true positive and less false positive peptides than PeptideProphet. Conclusions The proposed iterative method implemented according to the feedback framework can unify and improve the results of peptide identification and protein inference.

  12. vProtein: identifying optimal amino acid complements from plant-based foods.

    Directory of Open Access Journals (Sweden)

    Peter J Woolf

    Full Text Available BACKGROUND: Indispensible amino acids (IAAs are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. METHODS: vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. RESULTS: For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online

  13. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  14. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2015-08-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  15. Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

    Science.gov (United States)

    Zheng, Wu; Blake, Catherine

    2015-10-01

    Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    DEFF Research Database (Denmark)

    Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...

  17. Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis.

    Science.gov (United States)

    Bruneau, J M; Magnin, T; Tagat, E; Legrand, R; Bernard, M; Diaquin, M; Fudali, C; Latgé, J P

    2001-08-01

    Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W. A., Diaquin M., Popolo L., Hartland R. P., Latgé J.-P, J. Biol. Chem. 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane. To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A. fumigatus and the protein data were matched with the yeast genomic data. GPI-anchored proteins of A. fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis. They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing. Nine GPI-anchored proteins were identified in A. fumigatus. Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis. In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A. fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins. These results suggest that GPI-anchored proteins identified in this study are involved in A. fumigatus cell wall organization.

  18. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit

    2015-04-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  19. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit; Zhang, Huoming; Liu, Pei; Tsao, George Sai-wah; Li Lung, Maria; Mak, Nai-ki; Ngok-shun Wong, Ricky; Ying-kit Yue, Patrick

    2015-01-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  20. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses.

    Science.gov (United States)

    Eng, Christine L P; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak.

  1. A cohort of new adhesive proteins identified from transcriptomic analysis of mussel foot glands.

    Science.gov (United States)

    DeMartini, Daniel G; Errico, John M; Sjoestroem, Sebastian; Fenster, April; Waite, J Herbert

    2017-06-01

    The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion. © 2017 The Author(s).

  2. Hidden Markov model approach for identifying the modular framework of the protein backbone.

    Science.gov (United States)

    Camproux, A C; Tuffery, P; Chevrolat, J P; Boisvieux, J F; Hazout, S

    1999-12-01

    The hidden Markov model (HMM) was used to identify recurrent short 3D structural building blocks (SBBs) describing protein backbones, independently of any a priori knowledge. Polypeptide chains are decomposed into a series of short segments defined by their inter-alpha-carbon distances. Basically, the model takes into account the sequentiality of the observed segments and assumes that each one corresponds to one of several possible SBBs. Fitting the model to a database of non-redundant proteins allowed us to decode proteins in terms of 12 distinct SBBs with different roles in protein structure. Some SBBs correspond to classical regular secondary structures. Others correspond to a significant subdivision of their bounding regions previously considered to be a single pattern. The major contribution of the HMM is that this model implicitly takes into account the sequential connections between SBBs and thus describes the most probable pathways by which the blocks are connected to form the framework of the protein structures. Validation of the SBBs code was performed by extracting SBB series repeated in recoding proteins and examining their structural similarities. Preliminary results on the sequence specificity of SBBs suggest promising perspectives for the prediction of SBBs or series of SBBs from the protein sequences.

  3. Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

    DEFF Research Database (Denmark)

    Hauser, Frank; Williamson, Michael; Cazzamali, Giuseppe

    2006-01-01

    insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate...... receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships. Udgivelsesdato: 2006-Feb...

  4. Maize MeJA-responsive proteins identified by high-resolution 2-DE PAGE

    Directory of Open Access Journals (Sweden)

    Yuliang Zhang

    2015-12-01

    Full Text Available Exogenous methyl jasmonate (MeJA is well-known to induce plant defense mechanisms effective against a wide variety of insect and microbial pests. High-resolution 2-DE gel electrophoresis was used to discover changes in the leaf proteome of maize exposed to MeJA. We sequenced 62 MeJA-responsive proteins by tandem mass spectroscopy, and deposited the mass spectra and identities in the EMBL-EBI PRIDE repository under reference number PXD001793. An analysis and discussion of the identified proteins in relation to maize defense against Asian corn borer is published by Zhang et al. (2015 [1].

  5. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

  6. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  7. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    Science.gov (United States)

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  8. Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product.

    Science.gov (United States)

    Matsumoto, Takayuki; Hess, Sonja; Kajiyama, Hiroshi; Sakairi, Toru; Saleem, Moin A; Mathieson, Peter W; Nojima, Yoshihisa; Kopp, Jeffrey B

    2010-10-01

    The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of factor-binding protein-related protein-1 (IGFBP-rP1), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-β1 stimulation increased IGFBP-rP1 in conditioned medium. We analyzed IGFBP-rP1 glomerular expression in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteome may identify biomarkers of podocyte injury.

  9. Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

    DEFF Research Database (Denmark)

    Leon, Ileana R; Schwämmle, Veit; Jensen, Ole N

    2013-01-01

    a combination of qualitative and quantitative LC-MS/MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein...... conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents prior to analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative LC-MS/MS workflow quantified over 3700 distinct peptides with 96% completeness between all...... protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows...

  10. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing...... interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330...

  11. Effectively identifying compound-protein interactions by learning from positive and unlabeled examples.

    Science.gov (United States)

    Cheng, Zhanzhan; Zhou, Shuigeng; Wang, Yang; Liu, Hui; Guan, Jihong; Chen, Yi-Ping Phoebe

    2016-05-18

    Prediction of compound-protein interactions (CPIs) is to find new compound-protein pairs where a protein is targeted by at least a compound, which is a crucial step in new drug design. Currently, a number of machine learning based methods have been developed to predict new CPIs in the literature. However, as there is not yet any publicly available set of validated negative CPIs, most existing machine learning based approaches use the unknown interactions (not validated CPIs) selected randomly as the negative examples to train classifiers for predicting new CPIs. Obviously, this is not quite reasonable and unavoidably impacts the CPI prediction performance. In this paper, we simply take the unknown CPIs as unlabeled examples, and propose a new method called PUCPI (the abbreviation of PU learning for Compound-Protein Interaction identification) that employs biased-SVM (Support Vector Machine) to predict CPIs using only positive and unlabeled examples. PU learning is a class of learning methods that leans from positive and unlabeled (PU) samples. To the best of our knowledge, this is the first work that identifies CPIs using only positive and unlabeled examples. We first collect known CPIs as positive examples and then randomly select compound-protein pairs not in the positive set as unlabeled examples. For each CPI/compound-protein pair, we extract protein domains as protein features and compound substructures as chemical features, then take the tensor product of the corresponding compound features and protein features as the feature vector of the CPI/compound-protein pair. After that, biased-SVM is employed to train classifiers on different datasets of CPIs and compound-protein pairs. Experiments over various datasets show that our method outperforms six typical classifiers, including random forest, L1- and L2-regularized logistic regression, naive Bayes, SVM and k-nearest neighbor (kNN), and three types of existing CPI prediction models. Source code, datasets and

  12. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2011-01-01

    Full Text Available Abstract Background Enzymes in the radical SAM (rSAM domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses. Results An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ. Partial Phylogenetic Profiling (PPP based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P, but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such

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

    Energy Technology Data Exchange (ETDEWEB)

    Summers, Anne O. [Univ. of Georgia, Athens, GA (United States). Dept. of Microbiology; Miller, Susan M. [Univ. of California, San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry; Wall, Judy [Univ. of Missouri, Columbia, MO (United States). Dept. of Biochemistry; Lipton, Mary [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-18

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

  14. Identify High-Quality Protein Structural Models by Enhanced K-Means.

    Science.gov (United States)

    Wu, Hongjie; Li, Haiou; Jiang, Min; Chen, Cheng; Lv, Qiang; Wu, Chuang

    2017-01-01

    Background. One critical issue in protein three-dimensional structure prediction using either ab initio or comparative modeling involves identification of high-quality protein structural models from generated decoys. Currently, clustering algorithms are widely used to identify near-native models; however, their performance is dependent upon different conformational decoys, and, for some algorithms, the accuracy declines when the decoy population increases. Results. Here, we proposed two enhanced K -means clustering algorithms capable of robustly identifying high-quality protein structural models. The first one employs the clustering algorithm SPICKER to determine the initial centroids for basic K -means clustering ( SK -means), whereas the other employs squared distance to optimize the initial centroids ( K -means++). Our results showed that SK -means and K -means++ were more robust as compared with SPICKER alone, detecting 33 (59%) and 42 (75%) of 56 targets, respectively, with template modeling scores better than or equal to those of SPICKER. Conclusions. We observed that the classic K -means algorithm showed a similar performance to that of SPICKER, which is a widely used algorithm for protein-structure identification. Both SK -means and K -means++ demonstrated substantial improvements relative to results from SPICKER and classical K -means.

  15. Proteomics approach to identify unique xylem sap proteins in Pierce's disease-tolerant Vitis species.

    Science.gov (United States)

    Basha, Sheikh M; Mazhar, Hifza; Vasanthaiah, Hemanth K N

    2010-03-01

    Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

  16. Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins

    Science.gov (United States)

    2013-01-01

    Background Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation. PMID:24010718

  17. A newly identified protein of Leptospira interrogans mediates binding to laminin.

    Science.gov (United States)

    Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2009-10-01

    Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

  18. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    Directory of Open Access Journals (Sweden)

    Vladimir López

    2016-03-01

    Full Text Available Mycobacteria of the Mycobacterium tuberculosis complex (MTBC greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB. In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB- and M. bovis-infected young (TB+ and adult animals with different infection status [TB lesions localized in the head (TB+ or affecting multiple organs (TB++]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to

  19. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    Science.gov (United States)

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  20. A genome-wide association study identifies protein quantitative trait loci (pQTLs.

    Directory of Open Access Journals (Sweden)

    David Melzer

    2008-05-01

    Full Text Available There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts - cis effects, and elsewhere in the genome - trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8x10(-57, CCL4L1 (p = 3.9x10(-21, IL18 (p = 6.8x10(-13, LPA (p = 4.4x10(-10, GGT1 (p = 1.5x10(-7, SHBG (p = 3.1x10(-7, CRP (p = 6.4x10(-6 and IL1RN (p = 7.3x10(-6 genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R, altered secretion rates of different sized proteins (LPA, variation in gene copy number (CCL4L1 and altered transcription (GGT1. We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha levels (p = 6.8x10(-40, but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis

  1. Pharmacological and protein profiling suggest venetoclax (ABT-199) as optimal partner with ibrutinib in chronic lymphocytic leukemia

    Science.gov (United States)

    Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A.; Wierda, William G.; Keating, Michael J.; Balakrishnan, Kumudha; Gandhi, Varsha

    2015-01-01

    Purpose Bruton’s tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Experimental design Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199) and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. Results The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted highly cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family anti-apoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Conclusions Our biological and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. PMID:25829398

  2. Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A; Wierda, William G; Keating, Michael J; Balakrishnan, Kumudha; Gandhi, Varsha

    2015-08-15

    Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. ©2015 American Association for Cancer Research.

  3. A Proteomic Screen Identified Stress-Induced Chaperone Proteins as Targets of Akt Phosphorylation in Mesangial Cells

    OpenAIRE

    Barati, Michelle T.; Rane, Madhavi J.; Klein, Jon B.; McLeish, Kenneth R.

    2006-01-01

    The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90α, Hsp90β, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphoryl...

  4. EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

    Directory of Open Access Journals (Sweden)

    Vandenberg Albert

    2011-06-01

    Full Text Available Abstract Background Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. Results A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs in their deduced open reading frames (ORFs. The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs, candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain, cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP domain containing protein, unlike CP proteins

  5. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita; Straschil, Ursula; Bateman, Alex; Bö hme, Ulrike; Cherevach, Inna; Gong, Peng; Pain, Arnab; Billker, Oliver

    2010-01-01

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  6. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  7. Identifying biological concepts from a protein-related corpus with a probabilistic topic model

    Directory of Open Access Journals (Sweden)

    Lu Xinghua

    2006-02-01

    Full Text Available Abstract Background Biomedical literature, e.g., MEDLINE, contains a wealth of knowledge regarding functions of proteins. Major recurring biological concepts within such text corpora represent the domains of this body of knowledge. The goal of this research is to identify the major biological topics/concepts from a corpus of protein-related MEDLINE© titles and abstracts by applying a probabilistic topic model. Results The latent Dirichlet allocation (LDA model was applied to the corpus. Based on the Bayesian model selection, 300 major topics were extracted from the corpus. The majority of identified topics/concepts was found to be semantically coherent and most represented biological objects or concepts. The identified topics/concepts were further mapped to the controlled vocabulary of the Gene Ontology (GO terms based on mutual information. Conclusion The major and recurring biological concepts within a collection of MEDLINE documents can be extracted by the LDA model. The identified topics/concepts provide parsimonious and semantically-enriched representation of the texts in a semantic space with reduced dimensionality and can be used to index text.

  8. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    Science.gov (United States)

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-05-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

  9. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

    Directory of Open Access Journals (Sweden)

    Yaw Shin Ooi

    Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

  10. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    Science.gov (United States)

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  11. Automated local bright feature image analysis of nuclear protein distribution identifies changes in tissue phenotype

    International Nuclear Information System (INIS)

    Knowles, David; Sudar, Damir; Bator, Carol; Bissell, Mina

    2006-01-01

    The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, the distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is an increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype, and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently-stained nuclear protein NuMA in different mammary phenotypes obtained using three-dimensional cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from three-dimensional confocal images. Prominent features of fluorescently-stained NuMA were detected using a novel local bright feature analysis technique, and their normalized spatial density calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features as non-neoplastic cells underwent phenotypically normal acinar morphogenesis. In contrast, we did not detect any reorganization of NuMA during the formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating non-neoplastic cells from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues

  12. Bioinformatics analysis identify novel OB fold protein coding genes in C. elegans.

    Directory of Open Access Journals (Sweden)

    Daryanaz Dargahi

    Full Text Available BACKGROUND: The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5-25%. Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n=46 compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n=344 or fruit fly D. melanogaster (n=84. Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies. METHODOLOGY/PRINCIPAL FINDINGS: This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans. CONCLUSIONS/SIGNIFICANCE: This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.

  13. Social Partners

    DEFF Research Database (Denmark)

    Tikkanen, Tarja; Hansen, Leif Emil; Guðmundsson, Bernharður

    2012-01-01

    based on a survey carried out in the Nordic countries in the regie of Nordic Council of Ministries the article deals with the role of social partners in senior and older workers policies and practises......based on a survey carried out in the Nordic countries in the regie of Nordic Council of Ministries the article deals with the role of social partners in senior and older workers policies and practises...

  14. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  15. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Directory of Open Access Journals (Sweden)

    Kari A Dilley

    Full Text Available Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV, and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV. Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR activation.

  16. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Science.gov (United States)

    Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

    2017-01-01

    Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

  17. The BridgeDb framework: standardized access to gene, protein and metabolite identifier mapping services

    Directory of Open Access Journals (Sweden)

    Hanspers Kristina

    2010-01-01

    Full Text Available Abstract Background Many complementary solutions are available for the identifier mapping problem. This creates an opportunity for bioinformatics tool developers. Tools can be made to flexibly support multiple mapping services or mapping services could be combined to get broader coverage. This approach requires an interface layer between tools and mapping services. Results Here we present BridgeDb, a software framework for gene, protein and metabolite identifier mapping. This framework provides a standardized interface layer through which bioinformatics tools can be connected to different identifier mapping services. This approach makes it easier for tool developers to support identifier mapping. Mapping services can be combined or merged to support multi-omics experiments or to integrate custom microarray annotations. BridgeDb provides its own ready-to-go mapping services, both in webservice and local database forms. However, the framework is intended for customization and adaptation to any identifier mapping service. BridgeDb has already been integrated into several bioinformatics applications. Conclusion By uncoupling bioinformatics tools from mapping services, BridgeDb improves capability and flexibility of those tools. All described software is open source and available at http://www.bridgedb.org.

  18. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    Directory of Open Access Journals (Sweden)

    Wouter Boomsma

    2016-02-01

    Full Text Available The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work

  19. The antibacterial protein lysozyme identified as the termite egg recognition pheromone.

    Directory of Open Access Journals (Sweden)

    Kenji Matsuura

    Full Text Available Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP, which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence

  20. Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

    Directory of Open Access Journals (Sweden)

    Anna C Llewellyn

    Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

  1. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    Science.gov (United States)

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  2. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

    Science.gov (United States)

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

    2014-07-01

    Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  3. Intimate partner violence.

    Science.gov (United States)

    Cronholm, Peter F; Fogarty, Colleen T; Ambuel, Bruce; Harrison, Suzanne Leonard

    2011-05-15

    Intimate partner violence is a common source of physical, psychological, and emotional morbidity. In the United States, approximately 1.5 million women and 834,700 men annually are raped and/or physically assaulted by an intimate partner. Women are more likely than men to be injured, sexually assaulted, or murdered by an intimate partner. Studies suggest that one in four women is at lifetime risk. Physicians can use therapeutic relationships with patients to identify intimate partner violence, make brief office interventions, offer continuity of care, and refer them for subspecialty and community-based evaluation, treatment, and advocacy. Primary care physicians are ideally positioned to work from a preventive framework and address at-risk behaviors. Strategies for identifying intimate partner violence include asking relevant questions in patient histories, screening during periodic health examinations, and case finding in patients with suggestive signs or symptoms. Discussion needs to occur confidentially. Physicians should be aware of increased child abuse risk and negative effects on children's health observed in families with intimate partner violence. Physicians also should be familiar with local and national resources available to these patients.

  4. PARTNER Project

    CERN Multimedia

    Ballantine, A; Dixon-Altaber, H; Dosanjh, M; Kuchina, L

    2011-01-01

    Hadrontherapy uses particle beams to treat tumours located near critical organs and tumours that respond poorly to conventional radiation therapy. It has become evident that there is an emerging need for reinforcing research in hadrontherapy and it is essential to train professionals in this rapidly developing field. PARTNER is a 4-year Marie Curie Training project funded by the European Commission with 5.6 million Euros aimed at the creation of the next generation of experts. Ten academic institutes and research centres and two leading companies are participating in PARTNER, that is coordinated by CERN, forming a unique multidisciplinary and multinational European network. The project offers research and training opportunities to 25 young biologists, engineers, physicians and physicists and is allowing them to actively develop modern techniques for treating cancer in close collaboration with leading European Institutions. For this purpose PARTNER relies on cutting edge research and technology development, ef...

  5. LigSearch: a knowledge-based web server to identify likely ligands for a protein target

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Tjaart A. P. de; Laskowski, Roman A. [European Bioinformatics Institute (EMBL–EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Duban, Mark-Eugene [Northwestern University Feinberg School of Medicine, Chicago, Illinois (United States); Chan, A. W. Edith [University College London, London WC1E 6BT (United Kingdom); Anderson, Wayne F. [Northwestern University Feinberg School of Medicine, Chicago, Illinois (United States); Thornton, Janet M., E-mail: thornton@ebi.ac.uk [European Bioinformatics Institute (EMBL–EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom)

    2013-12-01

    LigSearch is a web server for identifying ligands likely to bind to a given protein. Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.

  6. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-telomeric Roles of Arabidopsis Telomerase

    Directory of Open Access Journals (Sweden)

    Ladislav eDokládal

    2015-11-01

    Full Text Available Telomerase-reverse transcriptase (TERT plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE TERT domain and identified a nuclear-localized protein that contains a RNA recognition motif (RRM. This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

  7. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

    Directory of Open Access Journals (Sweden)

    Aki Ieyasu

    2017-03-01

    Full Text Available Hematopoietic stem cells (HSCs are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

  8. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  9. Beyond BLASTing: Tertiary and Quaternary Structure Analysis Helps Identify Major Vault Proteins

    Science.gov (United States)

    Daly, Toni K.; Sutherland-Smith, Andrew J.; Penny, David

    2013-01-01

    We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via

  10. Y-box protein-1/p18 fragment identifies malignancies in patients with chronic liver disease

    International Nuclear Information System (INIS)

    Tacke, Frank; Kanig, Nicolas; En-Nia, Abdelaziz; Kaehne, Thilo; Eberhardt, Christiane S; Shpacovitch, Victoria; Trautwein, Christian; Mertens, Peter R

    2011-01-01

    Immunohistochemical detection of cold shock proteins is predictive for deleterious outcome in various malignant diseases. We recently described active secretion of a family member, denoted Y-box (YB) protein-1. We tested the clinical and diagnostic value of YB-1 protein fragment p18 (YB-1/p18) detection in blood for malignant diseases. We used a novel monoclonal anti-YB-1 antibody to detect YB-1/p18 by immunoblotting in plasma samples of healthy volunteers (n = 33), patients with non-cancerous, mostly inflammatory diseases (n = 60), hepatocellular carcinoma (HCC; n = 25) and advanced solid tumors (n = 20). YB-1/p18 was then tested in 111 patients with chronic liver diseases, alongside established tumor markers and various diagnostic measures, during evaluation for potential liver transplantation. We developed a novel immunoblot to detect the 18 kD fragment of secreted YB-1 in human plasma (YB-1/p18) that contains the cold-shock domains (CSD) 1-3 of the full-length protein. YB-1/p18 was detected in 11/25 HCC and 16/20 advanced carcinomas compared to 0/33 healthy volunteers and 10/60 patients with non-cancerous diseases. In 111 patients with chronic liver disease, YB-1/p18 was detected in 20 samples. Its occurrence was not associated with advanced Child stages of liver cirrhosis or liver function. In this cohort, YB-1/p18 was not a good marker for HCC, but proved most powerful in detecting malignancies other than HCC (60% positive) with a lower rate of false-positive results compared to established tumor markers. Alpha-fetoprotein (AFP) was most sensitive in detecting HCC, but simultaneous assessment of AFP, CA19-9 and YB-1/p18 improved overall identification of HCC patients. Plasma YB-1/p18 can identify patients with malignancies, independent of acute inflammation, renal impairment or liver dysfunction. The detection of YB-1/p18 in human plasma may have potential as a tumor marker for screening of high-risk populations, e.g. before organ transplantation, and should

  11. Functional characterisation of the Schizosaccharomyces pombe homologue of the leukaemia-associated translocation breakpoint binding protein translin and its binding partner, TRAX.

    Science.gov (United States)

    Jaendling, Alessa; Ramayah, Soshila; Pryce, David W; McFarlane, Ramsay J

    2008-02-01

    Translin is a conserved protein which associates with the breakpoint junctions of chromosomal translocations linked with the development of some human cancers. It binds to both DNA and RNA and has been implicated in mRNA metabolism and regulation of genome stability. It has a binding partner, translin-associated protein X (TRAX), levels of which are regulated by the translin protein in higher eukaryotes. In this study we find that this regulatory function is conserved in the lower eukaryotes, suggesting that translin and TRAX have important functions which provide a selective advantage to both unicellular and multi-cellular eukaryotes, indicating that this function may not be tissue-specific in nature. However, to date, the biological importance of translin and TRAX remains unclear. Here we systematically investigate proposals that suggest translin and TRAX play roles in controlling mitotic cell proliferation, DNA damage responses, genome stability, meiotic/mitotic recombination and stability of GT-rich repeat sequences. We find no evidence for translin and/or TRAX primary function in these pathways, indicating that the conserved biochemical function of translin is not implicated in primary pathways for regulating genome stability and/or segregation.

  12. Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

    Directory of Open Access Journals (Sweden)

    Michelle P Christie

    Full Text Available The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

  13. Beyond genes, proteins, and abstracts: Identifying scientific claims from full-text biomedical articles.

    Science.gov (United States)

    Blake, Catherine

    2010-04-01

    Massive increases in electronically available text have spurred a variety of natural language processing methods to automatically identify relationships from text; however, existing annotated collections comprise only bioinformatics (gene-protein) or clinical informatics (treatment-disease) relationships. This paper introduces the Claim Framework that reflects how authors across biomedical spectrum communicate findings in empirical studies. The Framework captures different levels of evidence by differentiating between explicit and implicit claims, and by capturing under-specified claims such as correlations, comparisons, and observations. The results from 29 full-text articles show that authors report fewer than 7.84% of scientific claims in an abstract, thus revealing the urgent need for text mining systems to consider the full-text of an article rather than just the abstract. The results also show that authors typically report explicit claims (77.12%) rather than an observations (9.23%), correlations (5.39%), comparisons (5.11%) or implicit claims (2.7%). Informed by the initial manual annotations, we introduce an automated approach that uses syntax and semantics to identify explicit claims automatically and measure the degree to which each feature contributes to the overall precision and recall. Results show that a combination of semantics and syntax is required to achieve the best system performance. 2009 Elsevier Inc. All rights reserved.

  14. Pleiotropy among common genetic loci identified for cardiometabolic disorders and C-reactive protein.

    Directory of Open Access Journals (Sweden)

    Symen Ligthart

    Full Text Available Pleiotropic genetic variants have independent effects on different phenotypes. C-reactive protein (CRP is associated with several cardiometabolic phenotypes. Shared genetic backgrounds may partially underlie these associations. We conducted a genome-wide analysis to identify the shared genetic background of inflammation and cardiometabolic phenotypes using published genome-wide association studies (GWAS. We also evaluated whether the pleiotropic effects of such loci were biological or mediated in nature. First, we examined whether 283 common variants identified for 10 cardiometabolic phenotypes in GWAS are associated with CRP level. Second, we tested whether 18 variants identified for serum CRP are associated with 10 cardiometabolic phenotypes. We used a Bonferroni corrected p-value of 1.1×10-04 (0.05/463 as a threshold of significance. We evaluated the independent pleiotropic effect on both phenotypes using individual level data from the Women Genome Health Study. Evaluating the genetic overlap between inflammation and cardiometabolic phenotypes, we found 13 pleiotropic regions. Additional analyses showed that 6 regions (APOC1, HNF1A, IL6R, PPP1R3B, HNF4A and IL1F10 appeared to have a pleiotropic effect on CRP independent of the effects on the cardiometabolic phenotypes. These included loci where individuals carrying the risk allele for CRP encounter higher lipid levels and risk of type 2 diabetes. In addition, 5 regions (GCKR, PABPC4, BCL7B, FTO and TMEM18 had an effect on CRP largely mediated through the cardiometabolic phenotypes. In conclusion, our results show genetic pleiotropy among inflammation and cardiometabolic phenotypes. In addition to reverse causation, our data suggests that pleiotropic genetic variants partially underlie the association between CRP and cardiometabolic phenotypes.

  15. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  16. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  17. Molecular characterization and functional analysis of PR-1-like proteins identified from the wheat head blight fungus Fusarium graminearum

    Science.gov (United States)

    The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologues are also found in other eukaryotic kingdoms. Studies on non-plant PR-1-like (PR-1L) proteins have been pursued widely in humans/animals but rarely in filamentous ascomycetes. Here we report the ch...

  18. Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo

    DEFF Research Database (Denmark)

    Horn, Signe; Kirkegaard, Jeannette S.; Hoelper, Soraya

    2016-01-01

    to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated...

  19. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

    DEFF Research Database (Denmark)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J

    2017-01-01

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

  20. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Directory of Open Access Journals (Sweden)

    Sae Tanaka

    Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  1. Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action.

    Science.gov (United States)

    Deng, Youping; Bhattacharya, Sujoy; Swamy, O Rama; Tandon, Ruchi; Wang, Yong; Janda, Robert; Riedel, Heimo

    2003-10-10

    The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling

  2. Ankle brachial index, C-reactive protein, and central augmentation index to identify individuals with severe atherosclerosis

    DEFF Research Database (Denmark)

    Eldrup, Nikolaj; Sillesen, Henrik; Prescott, Eva

    2006-01-01

    We examined the ability of ankle brachial index, C-reactive protein and central augmentation index to identify individuals in the general population with severe atherosclerosis, diagnosed as those with ischaemic cardiovascular disease.......We examined the ability of ankle brachial index, C-reactive protein and central augmentation index to identify individuals in the general population with severe atherosclerosis, diagnosed as those with ischaemic cardiovascular disease....

  3. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

    2009-01-01

    The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane...... purification and comparative quantitative LC-MS/MS proteomic analysis identified 13 membrane proteins that were expressed at higher levels and 3 that were under-expressed in the metastatic compared to the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5...

  4. Sequence Analysis of Hypothetical Proteins from 26695 to Identify Potential Virulence Factors

    Directory of Open Access Journals (Sweden)

    Ahmad Abu Turab Naqvi

    2016-09-01

    Full Text Available Helicobacter pylori is a Gram-negative bacteria that is responsible for gastritis in human. Its spiral flagellated body helps in locomotion and colonization in the host environment. It is capable of living in the highly acidic environment of the stomach with the help of acid adaptive genes. The genome of H. pylori 26695 strain contains 1,555 coding genes that encode 1,445 proteins. Out of these, 340 proteins are characterized as hypothetical proteins (HP. This study involves extensive analysis of the HPs using an established pipeline which comprises various bioinformatics tools and databases to find out probable functions of the HPs and identification of virulence factors. After extensive analysis of all the 340 HPs, we found that 104 HPs are showing characteristic similarities with the proteins with known functions. Thus, on the basis of such similarities, we assigned probable functions to 104 HPs with high confidence and precision. All the predicted HPs contain representative members of diverse functional classes of proteins such as enzymes, transporters, binding proteins, regulatory proteins, proteins involved in cellular processes and other proteins with miscellaneous functions. Therefore, we classified 104 HPs into aforementioned functional groups. During the virulence factors analysis of the HPs, we found 11 HPs are showing significant virulence. The identification of virulence proteins with the help their predicted functions may pave the way for drug target estimation and development of effective drug to counter the activity of that protein.

  5. Chagas disease vector blood meal sources identified by protein mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Judith I Keller

    Full Text Available Chagas disease is a complex vector borne parasitic disease involving blood feeding Triatominae (Hemiptera: Reduviidae insects, also known as kissing bugs, and the vertebrates they feed on. This disease has tremendous impacts on millions of people and is a global health problem. The etiological agent of Chagas disease, Trypanosoma cruzi (Kinetoplastea: Trypanosomatida: Trypanosomatidae, is deposited on the mammalian host in the insect's feces during a blood meal, and enters the host's blood stream through mucous membranes or a break in the skin. Identifying the blood meal sources of triatomine vectors is critical in understanding Chagas disease transmission dynamics, can lead to identification of other vertebrates important in the transmission cycle, and aids management decisions. The latter is particularly important as there is little in the way of effective therapeutics for Chagas disease. Several techniques, mostly DNA-based, are available for blood meal identification. However, further methods are needed, particularly when sample conditions lead to low-quality DNA or to assess the risk of human cross-contamination. We demonstrate a proteomics-based approach, using liquid chromatography tandem mass spectrometry (LC-MS/MS to identify host-specific hemoglobin peptides for blood meal identification in mouse blood control samples and apply LC-MS/MS for the first time to Triatoma dimidiata insect vectors, tracing blood sources to species. In contrast to most proteins, hemoglobin, stabilized by iron, is incredibly stable even being preserved through geologic time. We compared blood stored with and without an anticoagulant and examined field-collected insect specimens stored in suboptimal conditions such as at room temperature for long periods of time. To our knowledge, this is the first study using LC-MS/MS on field-collected arthropod disease vectors to identify blood meal composition, and where blood meal identification was confirmed with more

  6. Geomfinder: a multi-feature identifier of similar three-dimensional protein patterns: a ligand-independent approach.

    Science.gov (United States)

    Núñez-Vivanco, Gabriel; Valdés-Jiménez, Alejandro; Besoaín, Felipe; Reyes-Parada, Miguel

    2016-01-01

    Since the structure of proteins is more conserved than the sequence, the identification of conserved three-dimensional (3D) patterns among a set of proteins, can be important for protein function prediction, protein clustering, drug discovery and the establishment of evolutionary relationships. Thus, several computational applications to identify, describe and compare 3D patterns (or motifs) have been developed. Often, these tools consider a 3D pattern as that described by the residues surrounding co-crystallized/docked ligands available from X-ray crystal structures or homology models. Nevertheless, many of the protein structures stored in public databases do not provide information about the location and characteristics of ligand binding sites and/or other important 3D patterns such as allosteric sites, enzyme-cofactor interaction motifs, etc. This makes necessary the development of new ligand-independent methods to search and compare 3D patterns in all available protein structures. Here we introduce Geomfinder, an intuitive, flexible, alignment-free and ligand-independent web server for detailed estimation of similarities between all pairs of 3D patterns detected in any two given protein structures. We used around 1100 protein structures to form pairs of proteins which were assessed with Geomfinder. In these analyses each protein was considered in only one pair (e.g. in a subset of 100 different proteins, 50 pairs of proteins can be defined). Thus: (a) Geomfinder detected identical pairs of 3D patterns in a series of monoamine oxidase-B structures, which corresponded to the effectively similar ligand binding sites at these proteins; (b) we identified structural similarities among pairs of protein structures which are targets of compounds such as acarbose, benzamidine, adenosine triphosphate and pyridoxal phosphate; these similar 3D patterns are not detected using sequence-based methods; (c) the detailed evaluation of three specific cases showed the versatility

  7. New technique of identifying the hierarchy of dynamic domains in proteins using a method of molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    Yesylevskyy S. O.

    2010-04-01

    Full Text Available Aim. Despite a large number of existing domain identification techniques there is no universally accepted method, which identifies the hierarchy of dynamic domains using the data of molecular dynamics (MD simulations. The goal of this work is to develop such technique. Methods. The dynamic domains are identified by eliminating systematic motions from MD trajectories recursively in a model-free manner. Results. The technique called the Hierarchical Domain-Wise Alignment (HDWA to identify hierarchically organized dynamic domains in proteins using the MD trajectories has been developed. Conclusion. A new method of domain identification in proteins is proposed

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

    Science.gov (United States)

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

    2016-05-01

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

  9. Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

    Directory of Open Access Journals (Sweden)

    Boucher Charles AB

    2010-07-01

    Full Text Available Abstract Background The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics. Results Our analyses show that human proteins interacting with HIV form a densely connected and central sub-network within the total human protein interaction network. The evaluation of this sub-network for connectivity and centrality resulted in a set of proteins essential for the HIV life-cycle. Remarkably, we were able to associate proteins involved in RNA polymerase II transcription with hubs and proteasome formation with bottlenecks. Inferred network motifs show significant over-representation of positive and negative feedback patterns between virus and host. Strikingly, such patterns have never been reported in combined virus-host systems. Conclusions HIV infection results in a reprioritization of cellular processes reflected by an increase in the relative importance of transcriptional machinery and proteasome formation. We conclude that during the evolution of HIV, some patterns of interaction have been selected for resulting in a system where virus proteins preferably interact with central human proteins for direct control and with proteasomal proteins for indirect control over the cellular processes. Finally, the patterns described by network motifs illustrate how virus and host interact with one another.

  10. Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

    Science.gov (United States)

    Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

    2013-01-01

    Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

  11. Social Partners

    DEFF Research Database (Denmark)

    Hansen, Leif Emil

    2011-01-01

    The purpose of the paper is to present findings from a new Nordic survey on social partners’ policy and practice in regards older workers. The goal of the survey was to find out to what extent the social partners have developed policies and outlined strategies, which explicitly address the demogr...... lifelong learning and career development to their senior members during their last 15-20 years in working life. In this issue the social partners can and should play an active role – indeed, a leading role if needed – among the other key actors in society....... the demographic change and promote opportunities for lifelong learning and career development among their senior members (45+). Workforce in the Nordic countries tend to be highly organised – especially the older workers. The social partners’ involvement in the discussion of sustainable society...... and the contribution of lifelong learning to the needs and potential of older workers is crucial, as the demographic situation already today, and in particular the one to be expected within the next about 40 years, is historically without a precedent. The idea of continuous learning and the need for a meaningful work...

  12. Identifying the Proteins that Mediate the Ionizing Radiation Resistance of Deinococcus Radiodurans R1

    Energy Technology Data Exchange (ETDEWEB)

    Battista, John R

    2010-02-22

    The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy.

  13. An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates.

    Science.gov (United States)

    Hawley, Robert G; Chen, Yuzhong; Riz, Irene; Zeng, Chen

    2012-05-04

    In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members. Using a generalized motif, we performed a genome-wide search for novel BH3-containing proteins in the NCBI Consensus Coding Sequence (CCDS) database. In addition to known pro-apoptotic BH3-only proteins, 197 proteins were recovered that satisfied the search criteria. These were categorized according to α-helical content and predictive binding to BCL-xL (encoded by BCL2L1) and MCL-1, two representative anti-apoptotic BCL2 family members, using position-specific scoring matrix models. Notably, the list is enriched for proteins associated with autophagy as well as a broad spectrum of cellular stress responses such as endoplasmic reticulum stress, oxidative stress, antiviral defense, and the DNA damage response. Several potential novel BH3-containing proteins are highlighted. In particular, the analysis strongly suggests that the apoptosis inhibitor and DNA damage response regulator, AVEN, which was originally isolated as a BCL-xL-interacting protein, is a functional BH3-only protein representing a distinct subclass of BCL2 family members.

  14. Protein quantitative trait locus study in obesity during weight-loss identifies a leptin regulator

    DEFF Research Database (Denmark)

    Carayol, Jérôme; Chabert, Christian; Di Cara, Alessandro

    2017-01-01

    of an organism. Proteome analysis especially can provide new insights into the molecular mechanisms of complex traits like obesity. The role of genetic variation in determining protein level variation has not been assessed in obesity. To address this, we design a large-scale protein quantitative trait locus (p...

  15. Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

    Science.gov (United States)

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-12-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the

  16. Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

    Science.gov (United States)

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-01-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional

  17. Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

    Science.gov (United States)

    Spreafico, Filippo; Bongarzone, Italia; Pizzamiglio, Sara; Magni, Ruben; Taverna, Elena; De Bortoli, Maida; Ciniselli, Chiara M; Barzanò, Elena; Biassoni, Veronica; Luchini, Alessandra; Liotta, Lance A; Zhou, Weidong; Signore, Michele; Verderio, Paolo; Massimino, Maura

    2017-07-11

    Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

  18. Solution Structure, Membrane Interactions, and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni*

    Science.gov (United States)

    Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J.; Daly, Norelle L.; Gobert, Geoffrey N.; Jones, Malcolm K.; Craik, David J.; Mulvenna, Jason

    2014-01-01

    The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument. PMID:24429291

  19. Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

    Science.gov (United States)

    Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J; Daly, Norelle L; Gobert, Geoffrey N; Jones, Malcolm K; Craik, David J; Mulvenna, Jason

    2014-03-07

    The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.

  20. Pennsylvania's partnering process

    International Nuclear Information System (INIS)

    Latham, J.W.

    1996-01-01

    Pennsylvania is committed to finding a site for a low-level radioactive waste (LLRW) disposal facility through an innovative voluntary process. The Pennsylvania Department of Environmental Protection (DEP) and Chem-Nuclear Systems, Inc. (CNSI) developed the Community Partnering Plan with extensive public participation. The Community Partnering Plan outlines a voluntary process that empowers municipalities to evaluate the advantages and disadvantages of hosting the facility. DEP and CNSI began developing the Community Partnering Plan in July 1995. Before then, CNSI was using a screening process prescribed by state law and regulations to find a location for the facility. So far, approximately 78 percent of the Commonwealth has been identified as disqualified as a site for the LLRW disposal facility. The siting effort will now focus on identifying volunteer host municipalities in the remaining 22 percent of the state. This combination of technical screening and voluntary consideration makes Pennsylvania's process unique. A volunteered site will have to meet the same tough requirements for protecting people and the environment as a site chosen through the screening process. Protection of public health and safety continues to be the foundation of the state's siting efforts. The Community Partnering Plan offers a window of opportunity. If Pennsylvania does not find volunteer municipalities with suitable sites by the end of 1997, it probably will return to a technical screening process

  1. Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

    Science.gov (United States)

    Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

    2014-09-01

    Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

    Science.gov (United States)

    Ho, Steven C L; Yang, Yuansheng

    2014-08-01

    Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

  3. Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

    OpenAIRE

    Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E.; Rice, Andrew P.

    2015-01-01

    By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 ...

  4. IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

    DEFF Research Database (Denmark)

    Sparre, T; Bjerre-Christensen, Ulla; Mose Larsen, P

    2002-01-01

    of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen......Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression...

  5. Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum.

    Science.gov (United States)

    Lu, Shunwen; Edwards, Michael C

    2018-04-01

    The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum-wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

  6. Comparative genome analysis to identify SNPs associated with high oleic acid and elevated protein content in soybean.

    Science.gov (United States)

    Kulkarni, Krishnanand P; Patil, Gunvant; Valliyodan, Babu; Vuong, Tri D; Shannon, J Grover; Nguyen, Henry T; Lee, Jeong-Dong

    2018-03-01

    The objective of this study was to determine the genetic relationship between the oleic acid and protein content. The genotypes having high oleic acid and elevated protein (HOEP) content were crossed with five elite lines having normal oleic acid and average protein (NOAP) content. The selected accessions were grown at six environments in three different locations and phenotyped for protein, oil, and fatty acid components. The mean protein content of parents, HOEP, and NOAP lines was 34.6%, 38%, and 34.9%, respectively. The oleic acid concentration of parents, HOEP, and NOAP lines was 21.7%, 80.5%, and 20.8%, respectively. The HOEP plants carried both FAD2-1A (S117N) and FAD2-1B (P137R) mutant alleles contributing to the high oleic acid phenotype. Comparative genome analysis using whole-genome resequencing data identified six genes having single nucleotide polymorphism (SNP) significantly associated with the traits analyzed. A single SNP in the putative gene Glyma.10G275800 was associated with the elevated protein content, and palmitic, oleic, and linoleic acids. The genes from the marker intervals of previously identified QTL did not carry SNPs associated with protein content and fatty acid composition in the lines used in this study, indicating that all the genes except Glyma.10G278000 may be the new genes associated with the respective traits.

  7. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

    NARCIS (Netherlands)

    Soni, Siddarth; Raaijmakers, Antonia J A; Raaijmakers, Linsey M; Damen, J Mirjam A; van Stuijvenberg, Leonie; Vos, Marc A; Heck, Albert J R; van Veen, AAB; Scholten, Arjen

    2016-01-01

    AIMS: Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies

  8. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Directory of Open Access Journals (Sweden)

    Guangqi Li

    Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  9. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

    Directory of Open Access Journals (Sweden)

    Siddarth Soni

    Full Text Available Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID. The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2, Nexilin (NEXN, Popeye-domain-containg-protein 2 (POPDC2 and thioredoxin-related-transmembrane-protein 2 (TMX2 and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

  10. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    OpenAIRE

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

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

    Science.gov (United States)

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

    2012-01-01

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

  12. A systems biology strategy to identify molecular mechanisms of action and protein indicators of traumatic brain injury.

    Science.gov (United States)

    Yu, Chenggang; Boutté, Angela; Yu, Xueping; Dutta, Bhaskar; Feala, Jacob D; Schmid, Kara; Dave, Jitendra; Tawa, Gregory J; Wallqvist, Anders; Reifman, Jaques

    2015-02-01

    The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

  13. Comparative Genomics and Disorder Prediction Identify Biologically Relevant SH3 Protein Interactions.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Protein interaction networks are an important part of the post-genomic effort to integrate a part-list view of the cell into system-level understanding. Using a set of 11 yeast genomes we show that combining comparative genomics and secondary structure information greatly increases consensus-based prediction of SH3 targets. Benchmarking of our method against positive and negative standards gave 83% accuracy with 26% coverage. The concept of an optimal divergence time for effective comparative genomics studies was analyzed, demonstrating that genomes of species that diverged very recently from Saccharomyces cerevisiae(S. mikatae, S. bayanus, and S. paradoxus, or a long time ago (Neurospora crassa and Schizosaccharomyces pombe, contain less information for accurate prediction of SH3 targets than species within the optimal divergence time proposed. We also show here that intrinsically disordered SH3 domain targets are more probable sites of interaction than equivalent sites within ordered regions. Our findings highlight several novel S. cerevisiae SH3 protein interactions, the value of selection of optimal divergence times in comparative genomics studies, and the importance of intrinsic disorder for protein interactions. Based on our results we propose novel roles for the S. cerevisiae proteins Abp1p in endocytosis and Hse1p in endosome protein sorting.

  14. Comparative genomics and disorder prediction identify biologically relevant SH3 protein interactions.

    Directory of Open Access Journals (Sweden)

    Pedro Beltrao

    2005-08-01

    Full Text Available Protein interaction networks are an important part of the post-genomic effort to integrate a part-list view of the cell into system-level understanding. Using a set of 11 yeast genomes we show that combining comparative genomics and secondary structure information greatly increases consensus-based prediction of SH3 targets. Benchmarking of our method against positive and negative standards gave 83% accuracy with 26% coverage. The concept of an optimal divergence time for effective comparative genomics studies was analyzed, demonstrating that genomes of species that diverged very recently from Saccharomyces cerevisiae(S. mikatae, S. bayanus, and S. paradoxus, or a long time ago (Neurospora crassa and Schizosaccharomyces pombe, contain less information for accurate prediction of SH3 targets than species within the optimal divergence time proposed. We also show here that intrinsically disordered SH3 domain targets are more probable sites of interaction than equivalent sites within ordered regions. Our findings highlight several novel S. cerevisiae SH3 protein interactions, the value of selection of optimal divergence times in comparative genomics studies, and the importance of intrinsic disorder for protein interactions. Based on our results we propose novel roles for the S. cerevisiae proteins Abp1p in endocytosis and Hse1p in endosome protein sorting.

  15. Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid

    Science.gov (United States)

    Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

    2012-01-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  16. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    Science.gov (United States)

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  17. Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

    2016-11-01

    Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

  18. Incorporating deep learning with convolutional neural networks and position specific scoring matrices for identifying electron transport proteins.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

    2017-09-05

    In several years, deep learning is a modern machine learning technique using in a variety of fields with state-of-the-art performance. Therefore, utilization of deep learning to enhance performance is also an important solution for current bioinformatics field. In this study, we try to use deep learning via convolutional neural networks and position specific scoring matrices to identify electron transport proteins, which is an important molecular function in transmembrane proteins. Our deep learning method can approach a precise model for identifying of electron transport proteins with achieved sensitivity of 80.3%, specificity of 94.4%, and accuracy of 92.3%, with MCC of 0.71 for independent dataset. The proposed technique can serve as a powerful tool for identifying electron transport proteins and can help biologists understand the function of the electron transport proteins. Moreover, this study provides a basis for further research that can enrich a field of applying deep learning in bioinformatics. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Identifiability study of the proteins degradation model, based on ADM1, using simultaneous batch experiments

    DEFF Research Database (Denmark)

    Flotats, X.; Palatsi, J.; Ahring, Birgitte Kiær

    2006-01-01

    are not inhibiting the hydrolysis process. The ADM1 model adequately expressed the consecutive steps of hydrolysis and acidogenesis, with estimated kinetic values corresponding to a fast acidogenesis and slower hydrolysis. The hydrolysis was found to be the rate limiting step of anaerobic degradation. Estimation...... of yield coefficients based on the relative initial slopes of VFA profiles obtained in a simple batch experiment produced satisfactory results. From the identification study, it was concluded that it is possible to determine univocally the related kinetic parameter values for protein degradation...... if the evolution of amino acids is measured in simultaneous batch experiments, with different initial protein and amino acids concentrations....

  20. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  1. Identifying the adaptive mechanism in globular proteins: Fluctuations in densely packed regions manipulate flexible parts

    Science.gov (United States)

    Yilmaz, Lutfu Safak; Atilgan, Ali Rana

    2000-09-01

    A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier-Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuations that are widely distributed over the native structure. These positional arrangements do not appear in a reciprocal relation between a perturbation and the associated response; the effect of a movement of residue i on residue j is not equal to that of the same movement of residue j on residue i.

  2. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  3. Proteomics approach to identify dehydration responsive nuclear proteins from chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Pandey, Aarti; Chakraborty, Subhra; Datta, Asis; Chakraborty, Niranjan

    2008-01-01

    Dehydration or water-deficit is one of the most important environmental stress factors that greatly influences plant growth and development and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. Mechanisms that operate signal perception, transduction, and downstream regulatory events provide valuable information about the underlying pathways involved in environmental stress responses. The nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. To gain a better understanding of dehydration response in plants, we have developed a comparative nuclear proteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water and the changes in the nuclear proteome were examined using two-dimensional gel electrophoresis. Approximately 205 protein spots were found to be differentially regulated under dehydration. Mass spectrometry analysis allowed the identification of 147 differentially expressed proteins, presumably involved in a variety of functions including gene transcription and replication, molecular chaperones, cell signaling, and chromatin remodeling. The dehydration responsive nuclear proteome of chickpea revealed a coordinated response, which involves both the regulatory as well as the functional proteins. This study, for the first time, provides an insight into the complex metabolic network operating in the nucleus during dehydration.

  4. Identifying reliable predictors of protein-energy malnutrition in hospitalized frail older adults. A prospective longitudinal study.

    Science.gov (United States)

    Sanson, Gianfranco; Bertocchi, Luca; Dal Bo, Eugenia; Di Pasquale, Carmen Luisa; Zanetti, Michela

    2018-03-07

    Decreased food intake is a risk factor for relevant complications (e.g. infections, pressure ulcers), longer hospital stays, higher readmission rates, greater health care costs and increased patient mortality, particularly in frail hospitalized older adults who are malnourished or at risk of malnutrition. Nurses are called to improve this criticality, starting from accurately identify patients for malnutrition at hospital admission and effectively monitoring their food intake. The primary aim was to identify reliable predictive indicators of reduced food intake at hospital admission. The secondary aims were to assess the adequacy of daily energy and protein intake and the impact of nutrient intake on patient outcomes. Prospective observational longitudinal study. Internal Medicine Ward of an Academic Teaching University Hospital. Acute older adults who were malnourished or at risk of malnutrition (Nutritional Risk Score-2002 ≥ 3, middle-upper arm circumference energy and protein intake was monitored during the first 5 days of hospital stay by a photographic method and compared to the daily energy and protein requirement calculated by specific equations. Data on anthropometry, inflammation/malnutrition laboratory data and body composition (phase angle calculated using bioelectrical impedance analysis) were collected. Eighty-one subjects (age 81.5 ± 11.5 years) were enrolled. Mean energy intake was 669.0 ± 573.9 kcal/day, and mean protein intake was 30.7 ± 25.8 g/day. Over 60% of patients ingested ≤50% of their calculated energy and protein requirements: these patients were older (p = 0.026), had a lower middle-upper arm circumference (p = 0.022) and total arm area (p = 0.038), a higher C-reactive protein/albumin ratio and Instant Nutritional Assessment score (p protein/albumin ratio, and impaired self-feeding at admission were independently associated with critically reduced energy and protein intake. Middle

  5. A Large-Scale Quantitative Proteomic Approach to Identifying Sulfur Mustard-Induced Protein Phosphorylation Cascades

    Science.gov (United States)

    2010-01-01

    snapshot of SM-induced toxicity. Over the past few years, innovations in systems biology and biotechnology have led to important advances in our under...perturbations. SILAC has been used to study tumor metastasis (3, 4), focal adhesion- associated proteins, growth factor signaling, and insulin regula- tion (5...stained with colloidal Coomassie blue. After it was destained, the gel lane was excised into six regions, and each region was cut into 1 mm cubes

  6. Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell.

    Science.gov (United States)

    Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

    2016-03-01

    The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

  8. Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of polerovirus (luteoviridae).

    Science.gov (United States)

    Yang, Xiaolong; Thannhauser, T W; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E; Gray, Stewart M

    2008-01-01

    Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F(2) progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F(2) genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

  9. Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (Luteoviridae)▿

    Science.gov (United States)

    Yang, Xiaolong; Thannhauser, T. W.; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E.; Gray, Stewart M.

    2008-01-01

    Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F2 progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission. PMID:17959668

  10. An Asymmetric Deuterium Labeling Strategy to Identify Interprotomer and Intraprotomer NOEs in Oligomeric Proteins

    International Nuclear Information System (INIS)

    Jasanoff, Alan

    1998-01-01

    A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs. In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers. The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks. NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions. Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing with trypsin to yield only protomers with the desired N- and C-termini. In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present. This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments. Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates

  11. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    International Nuclear Information System (INIS)

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-01-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH 3 CN and the tyramine then labeled with 125 I. Dilactitol- 125 I-tyramine (DLT) and inulin- 125 I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol) 2 -N-CH 2 -CH 2 -NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins

  12. Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation.

    Science.gov (United States)

    Lewallen, Daniel M; Sreelatha, Anju; Dharmarajan, Venkatasubramanian; Madoux, Franck; Chase, Peter; Griffin, Patrick R; Orth, Kim; Hodder, Peter; Thompson, Paul R

    2014-02-21

    Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki's ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

  13. Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Leticia Barboza Rocha

    2017-10-01

    Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

  14. Strategy to Identify and Test Putative Light-Sensitive Non-Opsin G-Protein-Coupled Receptors: A Case Study.

    Science.gov (United States)

    Faggionato, Davide; Serb, Jeanne M

    2017-08-01

    The rise of high-throughput RNA sequencing (RNA-seq) and de novo transcriptome assembly has had a transformative impact on how we identify and study genes in the phototransduction cascade of non-model organisms. But the advantage provided by the nearly automated annotation of RNA-seq transcriptomes may at the same time hinder the possibility for gene discovery and the discovery of new gene functions. For example, standard functional annotation based on domain homology to known protein families can only confirm group membership, not identify the emergence of new biochemical function. In this study, we show the importance of developing a strategy that circumvents the limitations of semiautomated annotation and apply this workflow to photosensitivity as a means to discover non-opsin photoreceptors. We hypothesize that non-opsin G-protein-coupled receptor (GPCR) proteins may have chromophore-binding lysines in locations that differ from opsin. Here, we provide the first case study describing non-opsin light-sensitive GPCRs based on tissue-specific RNA-seq data of the common bay scallop Argopecten irradians (Lamarck, 1819). Using a combination of sequence analysis and three-dimensional protein modeling, we identified two candidate proteins. We tested their photochemical properties and provide evidence showing that these two proteins incorporate 11-cis and/or all-trans retinal and react to light photochemically. Based on this case study, we demonstrate that there is potential for the discovery of new light-sensitive GPCRs, and we have developed a workflow that starts from RNA-seq assemblies to the discovery of new non-opsin, GPCR-based photopigments.

  15. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

    Directory of Open Access Journals (Sweden)

    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  16. IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

    Science.gov (United States)

    Xing, Lili; Sun, Lina; Liu, Shilin; Li, Xiaoni; Zhang, Libin; Yang, Hongsheng

    2017-09-01

    Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers. Copyright © 2017. Published by Elsevier Inc.

  17. Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.

    Science.gov (United States)

    Cottet, Sandra; Corthésy-Theulaz, Irène; Spertini, François; Corthésy, Blaise

    2002-09-13

    Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

  18. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Science.gov (United States)

    Zhang, Yang; Devries, Mark E; Skolnick, Jeffrey

    2006-02-01

    G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness

  19. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  20. Proteomic-based detection of a protein cluster dysregulated during cardiovascular development identifies biomarkers of congenital heart defects.

    Directory of Open Access Journals (Sweden)

    Anjali K Nath

    Full Text Available Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs. However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies.Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry (WNT16, ST14, Pcsk1, Jumonji, Morca2a, TRPC5, and others were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins (WNT16, ADAM15 and NOGO-A/B was demonstrated in an ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1 as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF samples from women carrying normal fetuses and those with CHDs.The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

  1. Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Hu, Rongkuan; Huffman, Kenneth E; Chu, Michael; Zhang, Yajie; Minna, John D; Yu, Yonghao

    2016-02-05

    Lung cancer is the leading cause of cancer-related deaths for men and women in the United States, with non-small cell lung cancer (NSCLC) representing 85% of all diagnoses. Late stage detection, metastatic disease and lack of actionable biomarkers contribute to the high mortality rate. Proteins in the extracellular space are known to be critically involved in regulating every stage of the pathogenesis of lung cancer. To investigate the mechanism by which secreted proteins contribute to the pathogenesis of NSCLC, we performed quantitative secretomic analysis of two isogenic NSCLC cell lines (NCI-H1993 and NCI-H2073) and an immortalized human bronchial epithelial cell line (HBEC3-KT) as control. H1993 was derived from a chemo-naïve metastatic tumor, while H2073 was derived from the primary tumor after etoposide/cisplatin therapy. From the conditioned media of these three cell lines, we identified and quantified 2713 proteins, including a series of proteins involved in regulating inflammatory response, programmed cell death and cell motion. Gene Ontology (GO) analysis indicates that a number of proteins overexpressed in H1993 media are involved in biological processes related to cancer metastasis, including cell motion, cell-cell adhesion and cell migration. RNA interference (RNAi)-mediated knock down of a number of these proteins, including SULT2B1, CEACAM5, SPRR3, AGR2, S100P, and S100A14, leads to dramatically reduced migration of these cells. In addition, meta-analysis of survival data indicates NSCLC patients whose tumors express higher levels of several of these secreted proteins, including SULT2B1, CEACAM5, SPRR3, S100P, and S100A14, have a worse prognosis. Collectively, our results provide a potential molecular link between deregulated secretome and NSCLC cell migration/metastasis. In addition, the identification of these aberrantly secreted proteins might facilitate the development of biomarkers for early detection of this devastating disease.

  2. Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

    Directory of Open Access Journals (Sweden)

    Jerry R Colca

    Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

  3. Identifying stabilizing key residues in proteins using interresidue interaction energy matrix

    Czech Academy of Sciences Publication Activity Database

    Biedermannová, Lada; Hobza, Pavel; Vondrášek, Jiří

    2008-01-01

    Roč. 72, č. 1 (2008), s. 402-413 ISSN 0887-3585 R&D Projects: GA ČR GA203/05/0009; GA ČR GA203/06/1727; GA ČR(CZ) GD203/05/H001; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : protein stabilisation * an-initio calculation * interaction energy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.419, year: 2008

  4. Penicillin-binding proteins of Escherichia coli identified with a 125I-derivative of ampicillin

    International Nuclear Information System (INIS)

    Schwarz, U.; Seeger, K.; Wengenmayer, F.; Strecker, H.

    1981-01-01

    Evaluation of the binding of β-lactam antibiotics to penicillin-binding proteins (PBPs) in the bacterial cell wall by the established method using 14 C-labelled penicillin G has some disadvantages. Due to the small number of PBP molecules and the relatively low specific activity of [ 14 C]penicillin G available, very long exposure times for autoradiography are required. Furthermore, additional radiolabelled derivatives of penicillin with modified binding patterns might reveal PBPs not known so far. The authors describe the synthesis of a 125 I-labelled derivative of ampicillin and the labelling of PBPs with this compound. (Auth.)

  5. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

    Science.gov (United States)

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T; Texier, Yves; van Beersum, Sylvia E C; Horn, Nicola; Willer, Jason R; Mans, Dorus A; Dougherty, Gerard; Lamers, Ideke J C; Coene, Karlien L M; Arts, Heleen H; Betts, Matthew J; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J F; Marcelis, Carlo L; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A; Toedt, Grischa; van Dam, Teunis J P; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L; Blacque, Oliver E; Gibson, Toby J; Huynen, Martijn A; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E; Franco, Brunella; Giles, Rachel H; Ueffing, Marius; Russell, Robert B; Roepman, Ronald

    2016-05-13

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.

  6. Patient preferences for partner notification.

    Science.gov (United States)

    Apoola, A; Radcliffe, K W; Das, S; Robshaw, V; Gilleran, G; Kumari, B S; Boothby, M; Rajakumar, R

    2006-08-01

    To identify patient preferences for notification of sexual contacts when a sexually transmitted infection (STI) is diagnosed. A questionnaire survey of 2544 patients attending three large genitourinary clinics at Derby, Birmingham, and Coventry in the United Kingdom. The median age of the respondents was 24 with 1474 (57.9%) women, 1835 (72.1%) white, 1826 (71.8%) single. The most favoured method of partner notification was patient referral, which was rated a "good" method by 65.8% when they had to be contacted because a sexual partner has an STI. Notifying contacts by letter as a method of provider partner notification is more acceptable than phoning, text messaging, or email. Respondents with access to mobile telephones, private emails, and private letters were more likely to rate a method of partner notification using that mode of communication as "good" compared to those without. With provider referral methods of partner notification respondents preferred to receive a letter, email, or text message asking them to contact the clinic rather than a letter, email or text message informing them that they may have an STI. Most respondents think that being informed directly by a partner is the best method of being notified of the risk of an STI. Some of the newer methods may not be acceptable to all but a significant minority of respondents prefer these methods of partner notification. The wording of letters, emails, or text messages when used for partner notification has an influence on the acceptability of the method and may influence success of the partner notification method. Services should be flexible enough to utilise the patients' preferred method of partner notification.

  7. Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes (F).

    Science.gov (United States)

    Bonilla, Martha L; Todd, Christopher; Erlandson, Martin; Andres, Jose

    2015-12-22

    Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies.

  8. Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy.

    Science.gov (United States)

    Li, Jiajia; Ding, Xianlong; Han, Shaohuai; He, Tingting; Zhang, Hao; Yang, Longshu; Yang, Shouping; Gai, Junyi

    2016-04-14

    To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Soybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean

  9. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Boomsma, Wouter Krogh; Nielsen, Sofie Vincents; Lindorff-Larsen, Kresten

    2016-01-01

    conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology...

  10. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins.

    Science.gov (United States)

    Macaulay, Iain C; Tijssen, Marloes R; Thijssen-Timmer, Daphne C; Gusnanto, Arief; Steward, Michael; Burns, Philippa; Langford, Cordelia F; Ellis, Peter D; Dudbridge, Frank; Zwaginga, Jaap-Jan; Watkins, Nicholas A; van der Schoot, C Ellen; Ouwehand, Willem H

    2007-04-15

    To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

  11. Proteomics identifies Bacillus cereus EntD as a pivotal protein for the production of numerous virulence factors

    Directory of Open Access Journals (Sweden)

    Hélène eOmer

    2015-10-01

    Full Text Available Bacillus cereus is a gram-positive pathogen that causes a wide variety of diseases in humans. It secretes into the extracellular milieu proteins that may contribute directly or indirectly to its virulence. EntD is a novel exoprotein identified by proteogenomics of B. cereus ATCC 14579. We constructed a ΔentD mutant and analyzed the impact of entD disruption on the cellular proteome and exoproteome isolated from early, late and stationary-phase cultures. We identified 308 and 79 proteins regulated by EntD in the cellular proteome and the exoproteome, respectively. The contribution of these proteins to important virulence-associated functions, including central metabolism, cell structure, antioxidative ability, cell motility and toxin production, are presented. The proteomic data were correlated with the growth defect, cell morphology change, reduced motility and reduced cytotoxicity of the ΔentD mutant strain. We conclude that EntD is an important player in B. cereus virulence. The function of EntD and the putative EntD-dependent regulatory network are discussed. To our knowledge, this study is the first characterization of an Ent family protein in a species of the B. cereus group.

  12. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration.

    Science.gov (United States)

    Chen, Hsin-Hsiung; Chen, Wen-Pin; Yan, Wan-Lun; Huang, Yuan-Chun; Chang, Szu-Wei; Fu, Wen-Mei; Su, Ming-Jai; Yu, I-Shing; Tsai, Tzung-Chieh; Yan, Yu-Ting; Tsao, Yeou-Ping; Chen, Show-Li

    2015-11-15

    Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity. © 2015. Published by The Company of Biologists Ltd.

  14. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Directory of Open Access Journals (Sweden)

    Guan-Feng Wang

    2015-02-01

    Full Text Available Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR. Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC, a nucleotide binding (NB-ARC and a leucine rich repeat (LRR domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  15. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Science.gov (United States)

    Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

    2015-02-01

    Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  16. Regional Differences of Proteins Expressing in Adipose Depots Isolated from Cows, Steers and Bulls as Identified by a Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Jin Hyoung Cho

    2016-08-01

    Full Text Available Adipose tissue in the loin muscle area of beef cattle as a marbling factor is directly associated with beef quality. To elucidate whether properties of proteins involved in depot specific adipose tissue were sex-dependent, we analyzed protein expression of intramuscular adipose tissue (IMAT and omental adipose tissue (OMAT from Hanwoo cows, steers, and bulls of Korean native beef cattle by liquid chromatography-tandem mass spectrometry (LC-MS/MS–based proteomic analysis, quantitative polymerase chain reaction (PCR and western blot analysis. Two different adipose depots (i.e. intramuscular and omental were collected from cows (n = 7, steers (n = 7, or bulls (n = 7. LC-MS/MS revealed a total of 55 and 35 proteins in IMAT and OMAT, respectively. Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively. Tropomyosin (TPM 1, TPM 2, and TPM3 were subjected to verification by quantitative PCR and western blot analysis in IMAT and OMAT of Hanwoo cows, steers, and bulls as key factors closely associated with muscle development. Both mRNA levels and protein levels of TPM1, TPM2, and TPM3 in IMAT were lower in bulls compared to in cows or steers suggesting that they were positively correlated with marbling score and quality grade. Our results may aid the regulation of marbling development and improvement of meat quality grades in beef cattle.

  17. FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

    Science.gov (United States)

    Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

    2008-06-12

    Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

  18. Multi-Population Selective Genotyping to Identify Soybean [Glycine max (L.) Merr.] Seed Protein and Oil QTLs.

    Science.gov (United States)

    Phansak, Piyaporn; Soonsuwon, Watcharin; Hyten, David L; Song, Qijian; Cregan, Perry B; Graef, George L; Specht, James E

    2016-06-01

    Plant breeders continually generate ever-higher yielding cultivars, but also want to improve seed constituent value, which is mainly protein and oil, in soybean [Glycine max (L.) Merr.]. Identification of genetic loci governing those two traits would facilitate that effort. Though genome-wide association offers one such approach, selective genotyping of multiple biparental populations offers a complementary alternative, and was evaluated here, using 48 F2:3 populations (n = ∼224 plants) created by mating 48 high protein germplasm accessions to cultivars of similar maturity, but with normal seed protein content. All F2:3 progeny were phenotyped for seed protein and oil, but only 22 high and 22 low extreme progeny in each F2:3 phenotypic distribution were genotyped with a 1536-SNP chip (ca 450 bimorphic SNPs detected per mating). A significant quantitative trait locus (QTL) on one or more chromosomes was detected for protein in 35 (73%), and for oil in 25 (52%), of the 48 matings, and these QTL exhibited additive effects of ≥ 4 g kg(-1) and R(2) values of 0.07 or more. These results demonstrated that a multiple-population selective genotyping strategy, when focused on matings between parental phenotype extremes, can be used successfully to identify germplasm accessions possessing large-effect QTL alleles. Such accessions would be of interest to breeders to serve as parental donors of those alleles in cultivar development programs, though 17 of the 48 accessions were not unique in terms of SNP genotype, indicating that diversity among high protein accessions in the germplasm collection is less than what might ordinarily be assumed. Copyright © 2016 Phansak et al.

  19. Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

    Science.gov (United States)

    Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

    2008-12-01

    Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

  20. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

    Science.gov (United States)

    Toret, Christopher P.; D’Ambrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

    2014-01-01

    Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

  1. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    Science.gov (United States)

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  2. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    Directory of Open Access Journals (Sweden)

    Chih-Yuan eChiang

    2015-07-01

    Full Text Available Burkholderia is a diverse genus of Gram-negative bacteria that cause high mortality rate in humans and cattle. The lack of effective therapeutic treatments poses serious public health threats. Insights toward host-Burkholderia spp. interaction are critical in understanding the pathogenesis of the infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray (RPMA technology was previously proven to characterize novel biomarkers and molecular signatures associated with infectious diseases and cancers. In the present study, this technology was utilized to interrogate changes in host protein expression and post-translational phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections and of which eight proteins were selected for further validation by immunoblotting. Kinetic expression patterns of phosphorylated AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune responses. Modulating inflammatory responses by perturbing AMPK-α1, Src, and GSK3β activities may provide novel therapeutic targets for future treatments.

  3. Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

    Science.gov (United States)

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-09-19

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

    Science.gov (United States)

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-01-01

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

  5. Features of two new proteins with OmpA-like domains identified in the genome sequences of Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Aline F Teixeira

    Full Text Available Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively. Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG-interacting proteins, capable of generating plasmin (PLA and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis.

  6. Metaproteomics of saliva identifies human protein markers specific for individuals with periodontitis and dental caries compared to orally healthy controls

    DEFF Research Database (Denmark)

    Belstrøm, Daniel; Jersie-Christensen, Rosa R; Lyon, David

    2016-01-01

    BACKGROUND: The composition of the salivary microbiota has been reported to differentiate between patients with periodontitis, dental caries and orally healthy individuals. To identify characteristics of diseased and healthy saliva we thus wanted to compare saliva metaproteomes from patients...... with periodontitis and dental caries to healthy individuals. METHODS: Stimulated saliva samples were collected from 10 patients with periodontitis, 10 patients with dental caries and 10 orally healthy individuals. The proteins in the saliva samples were subjected to denaturing buffer and digested enzymatically...... and inflammatory markers in periodontitis and dental caries compared to healthy controls. Bacterial proteome profiles and functional annotation were very similar in health and disease. CONCLUSIONS: Overexpression of proteins related to the complement system and inflammation seems to correlate with oral disease...

  7. Simple and efficient machine learning frameworks for identifying protein-protein interaction relevant articles and experimental methods used to study the interactions.

    Science.gov (United States)

    Agarwal, Shashank; Liu, Feifan; Yu, Hong

    2011-10-03

    Protein-protein interaction (PPI) is an important biomedical phenomenon. Automatically detecting PPI-relevant articles and identifying methods that are used to study PPI are important text mining tasks. In this study, we have explored domain independent features to develop two open source machine learning frameworks. One performs binary classification to determine whether the given article is PPI relevant or not, named "Simple Classifier", and the other one maps the PPI relevant articles with corresponding interaction method nodes in a standardized PSI-MI (Proteomics Standards Initiative-Molecular Interactions) ontology, named "OntoNorm". We evaluated our system in the context of BioCreative challenge competition using the standardized data set. Our systems are amongst the top systems reported by the organizers, attaining 60.8% F1-score for identifying relevant documents, and 52.3% F1-score for mapping articles to interaction method ontology. Our results show that domain-independent machine learning frameworks can perform competitively well at the tasks of detecting PPI relevant articles and identifying the methods that were used to study the interaction in such articles. Simple Classifier is available at http://sourceforge.net/p/simpleclassify/home/ and OntoNorm at http://sourceforge.net/p/ontonorm/home/.

  8. Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

    Science.gov (United States)

    Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

    2016-08-01

    Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function[OPEN

    Science.gov (United States)

    2018-01-01

    Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771

  10. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function.

    Science.gov (United States)

    Kourelis, Jiorgos; van der Hoorn, Renier A L

    2018-02-01

    Plants have many, highly variable resistance ( R ) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize ( Zea mays ) Hm1 , was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. © 2018 American Society of Plant Biologists. All rights reserved.

  11. Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host-pathogen interactions

    Directory of Open Access Journals (Sweden)

    Laura eGomez-Valero

    2011-10-01

    Full Text Available Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic-like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

  12. Genetic interactions of MAF1 identify a role for Med20 in transcriptional repression of ribosomal protein genes.

    Directory of Open Access Journals (Sweden)

    Ian M Willis

    2008-07-01

    Full Text Available Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription under multiple conditions. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis.

  13. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Science.gov (United States)

    Greub, Gilbert; Kebbi-Beghdadi, Carole; Bertelli, Claire; Collyn, François; Riederer, Beat M; Yersin, Camille; Croxatto, Antony; Raoult, Didier

    2009-12-23

    With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  14. ApicoAP: the first computational model for identifying apicoplast-targeted proteins in multiple species of Apicomplexa.

    Directory of Open Access Journals (Sweden)

    Gokcen Cilingir

    Full Text Available Most of the parasites of the phylum Apicomplexa contain a relict prokaryotic-derived plastid called the apicoplast. This organelle is important not only for the survival of the parasite, but its unique properties make it an ideal drug target. The majority of apicoplast-associated proteins are nuclear encoded and targeted post-translationally to the organellar lumen via a bipartite signaling mechanism that requires an N-terminal signal and transit peptide (TP. Attempts to define a consensus motif that universally identifies apicoplast TPs have failed.In this study, we propose a generalized rule-based classification model to identify apicoplast-targeted proteins (ApicoTPs that use a bipartite signaling mechanism. Given a training set specific to an organism, this model, called ApicoAP, incorporates a procedure based on a genetic algorithm to tailor a discriminating rule that exploits the known characteristics of ApicoTPs. Performance of ApicoAP is evaluated for four labeled datasets of Plasmodium falciparum, Plasmodium yoelii, Babesia bovis, and Toxoplasma gondii proteins. ApicoAP improves the classification accuracy of the published dataset for P. falciparum to 94%, originally 90% using PlasmoAP.We present a parametric model for ApicoTPs and a procedure to optimize the model parameters for a given training set. A major asset of this model is that it is customizable to different parasite genomes. The ApicoAP prediction software is available at http://code.google.com/p/apicoap/ and http://bcb.eecs.wsu.edu.

  15. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Philip W. Brownjohn

    2017-04-01

    Full Text Available Summary: Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP processing and the accumulation of APP-derived amyloid β (Aβ peptides are key processes in Alzheimer's disease (AD. We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation. : In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex. Keywords: neural stem cells, Alzheimer's disease, phenotypic screening, iPSCs, human neurons, dementia, Down syndrome, amyloid beta, ivermectin, selamectin

  16. New Partner Orientation

    Science.gov (United States)

    This EPA presentation provides information on the SmartWay Transport Partnership Program, including key information about EPA, Partners' roles, benefits, tools, partner recognition, awards, and brand value. Transcript available.

  17. Green Power Partner Resources

    Science.gov (United States)

    EPA Green Power Partners can access tools and resources to help promote their green power commitments. Partners use these tools to communicate the benefits of their green power use to their customers, stakeholders, and the general public.

  18. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  19. A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

    Science.gov (United States)

    Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro

    2018-02-01

    Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.

  20. Novel peptide marker corresponding to salivary protein gSG6 potentially identifies exposure to Anopheles bites.

    Directory of Open Access Journals (Sweden)

    Anne Poinsignon

    2008-06-01

    Full Text Available In order to improve malaria control, and under the aegis of WHO recommendations, many efforts are being devoted to developing new tools for identifying geographic areas with high risk of parasite transmission. Evaluation of the human antibody response to arthropod salivary proteins could be an epidemiological indicator of exposure to vector bites, and therefore to risk of pathogen transmission. In the case of malaria, which is transmitted only by anopheline mosquitoes, maximal specificity could be achieved through identification of immunogenic proteins specific to the Anopheles genus. The objective of the present study was to determine whether the IgG response to the Anopheles gambiae gSG6 protein, from its recombinant form to derived synthetic peptides, could be an immunological marker of exposure specific to Anopheles gambiae bites.Specific IgG antibodies to recombinant gSG6 protein were observed in children living in a Senegalese area exposed to malaria. With the objective of optimizing Anopheles specificity and reproducibility, we designed five gSG6-based peptide sequences using a bioinformatic approach, taking into consideration i their potential antigenic properties and ii the absence of cross-reactivity with protein sequences of other arthropods/organisms. The specific anti-peptide IgG antibody response was evaluated in exposed children. The five gSG6 peptides showed differing antigenic properties, with gSG6-P1 and gSG6-P2 exhibiting the highest antigenicity. However, a significant increase in the specific IgG response during the rainy season and a positive association between the IgG level and the level of exposure to Anopheles gambiae bites was significant only for gSG6-P1.This step-by-step approach suggests that gSG6-P1 could be an optimal candidate marker for evaluating exposure to Anopheles gambiae bites. This marker could be employed as a geographic indicator, like remote sensing techniques, for mapping the risk of malaria. It could

  1. Partnering and contracting

    DEFF Research Database (Denmark)

    Bohnstedt, Kristian Ditlev

    2014-01-01

    Purpose - Partnering is often, by economists, and construction managerial literature related to more incomplete contracts. This can be explained by seeing partnering as something that neutralizes opportunism. The aim is to uncover whether partnering neutralizes opportunism when there is an incomp...

  2. Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

    Science.gov (United States)

    Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

    2010-05-05

    Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  3. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    2010-05-01

    Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  4. Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

    Science.gov (United States)

    Isensee, Jörg; Wenzel, Carsten; Buschow, Rene; Weissmann, Robert; Kuss, Andreas W.; Hucho, Tim

    2014-01-01

    Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD2 activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD2 producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based on the elimination

  5. The Immunohistochemical Analysis of SOCS3 Protein Identifies a Subgroup of Prostatic Cancer Biopsies With Aggressive Behavior.

    Science.gov (United States)

    Pierconti, Francesco; Martini, Maurizio; Cenci, Tonia; Larocca, Luigi M

    Recently, we demonstrated that hypermethylation of SOCS3 determines a significant reduction of its mRNA and protein expression and identifies a subgroup of prostate cancer with aggressive behavior. In this paper, our objective was to investigate whether the immunohistochemical expression of the SOCS3 protein could represent an alternative method to molecular analysis for the individualization of aggressive prostate carcinoma. We analyzed the SOCS3 immunohistochemical expression in 65 patients undergoing biopsies at the Institute of Urology of our hospital between September 2011 and October 2011 (median age, 66.4 y; range, 50 to 73 y), and in 35 cases, a subset of 65 cases originally used for the immunohistochemical study, we studied the methylation status of the SOCS3 promoter. We found that the percentage of cases with SOCS3 negativity (-) or with SOCS3 weak staining in <50% of the neoplastic glands (+/-) correlated to the worst prognosis in terms of the Gleason score (P=0.0001; Fisher's exact test), the pT stage (P=0.012; Fisher's exact test), and progression-free survival (P=0.0334; hazard ratio, 0.34; and 95% confidence interval, from 0.1261 to 0.9188). Moreover, some cases with an SOCS3 unmethylated pattern showed SOCS3-negative immunostaining (-) or SOCS3-negative glands with weak cytoplasmatic staining in <50% of the neoplastic glands (+/-). Our data suggest that in prostatic cancer biopsies, the immunohistochemical analysis of SOCS3 protein expression may provide a method that is less expensive and easier to apply than SOCS3 methylation analysis for the distinction of a subgroup of prostate cancer with a more aggressive behavior.

  6. Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties

    KAUST Repository

    Lee, H.

    2010-02-26

    Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.

  7. High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines.

    Directory of Open Access Journals (Sweden)

    Ireos Filipuzzi

    2016-11-01

    Full Text Available Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.

  8. Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties

    KAUST Repository

    Lee, H.; Damsz, B.; Woloshuk, C. P.; Bressan, R. A.; Narasimhan, Meena L.

    2010-01-01

    Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.

  9. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Directory of Open Access Journals (Sweden)

    Gilbert Greub

    Full Text Available BACKGROUND: With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. METHODS/PRINCIPAL FINDINGS: We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. CONCLUSIONS/SIGNIFICANCE: This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  10. Pharma partnering: other people's science.

    Science.gov (United States)

    Hofmann, Christian

    2017-01-01

    Partnering is an ideal field if someone is seeking to move from a scientific to a more business-oriented discipline. Partnering's goal is to identify and acquire external innovation. These discoveries are then included in a company's pipeline and help bring novel treatments to patients. Advanced scientific training is essential in the identification and evaluation of these external assets. Here I describe how partnering works in a pharmaceutical company and offer advice on how to make a successful transition from a PhD program to a business career. © 2017 Hofmann. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  11. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Science.gov (United States)

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  12. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Directory of Open Access Journals (Sweden)

    Benoît de Chassey

    Full Text Available Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1 appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  13. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  14. Identification of sumoylation sites in CCDC6, the first identified RET partner gene in papillary thyroid carcinoma, uncovers a mode of regulating CCDC6 function on CREB1 transcriptional activity.

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    Chiara Luise

    Full Text Available CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424 which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.

  15. Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

    Directory of Open Access Journals (Sweden)

    Ying-Tzu Tseng

    Full Text Available Severe acute respiratory syndrome coronavirus (SARS-CoV membrane (M proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs when coexpressed with SARS-CoV nucleocapsid (N protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219 is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.

  16. A molecular key for building hyphae aggregates: the role of the newly identified Streptomyces protein HyaS.

    Science.gov (United States)

    Koebsch, Ilona; Overbeck, Jens; Piepmeyer, Sophie; Meschke, Holger; Schrempf, Hildgund

    2009-05-01

    Streptomycetes produce many metabolites with medical and biotechnological applications. During fermentations, their hyphae build aggregates, a process in which the newly identified protein HyaS plays an important role. The corresponding hyaS gene is present within all investigated Streptomyces species. Reporter fusions indicate that transcription of hyaS occurs within substrate hyphae of the Streptomyces lividans wild type (WT). The HyaS protein is dominantly associated with the substrate hyphae. The WT strain forms cylindrically shaped clumps of densely packed substrate hyphae, often fusing to higher aggregates (pellets), which remain stably associated during shaking. Investigations by electron microscopy suggest that HyaS induces tight fusion-like contacts among substrate hyphae. In contrast, the pellets of the designed hyaS disruption mutant ΔH are irregular in shape, contain frequently outgrowing bunches of hyphae, and fuse less frequently. ΔH complemented with a plasmid carrying hyaS resembles the WT phenotype. Biochemical studies indicate that the C-terminal region of HyaS has amine oxidase activity. Investigations of ΔH transformants, each carrying a specifically mutated gene, lead to the conclusion that the in situ oxidase activity correlates with the pellet-inducing role of HyaS, and depends on the presence of certain histidine residues. Furthermore, the level of undecylprodigiosin, a red pigment with antibiotic activity, is influenced by the engineered hyaS subtype within a strain. These data present the first molecular basis for future manipulation of pellets, and concomitant production of secondary metabolites during biotechnological processes. © 2009 The Authors. Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

  17. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  18. Comparative Genomics Identifies a Novel Conserved Protein, HpaT, in Proteobacterial Type III Secretion Systems that Do Not Possess the Putative Translocon Protein HrpF

    Directory of Open Access Journals (Sweden)

    Céline Pesce

    2017-06-01

    Full Text Available Xanthomonas translucens is the causal agent of bacterial leaf streak, the most common bacterial disease of wheat and barley. To cause disease, most xanthomonads depend on a highly conserved type III secretion system, which translocates type III effectors into host plant cells. Mutagenesis of the conserved type III secretion gene hrcT confirmed that the X. translucens type III secretion system is required to cause disease on the host plant barley and to trigger a non-host hypersensitive response (HR in pepper leaves. Type III effectors are delivered to the host cell by a surface appendage, the Hrp pilus, and a translocon protein complex that inserts into the plant cell plasma membrane. Homologs of the Xanthomonas HrpF protein, including PopF from Ralstonia solanacearum and NolX from rhizobia, are thought to act as a translocon protein. Comparative genomics revealed that X. translucens strains harbor a noncanonical hrp gene cluster, which rather shares features with type III secretion systems from Ralstonia solanacearum, Paraburkholderia andropogonis, Collimonas fungivorans, and Uliginosibacterium gangwonense than other Xanthomonas spp. Surprisingly, none of these bacteria, except R. solanacearum, encode a homolog of the HrpF translocon. Here, we aimed at identifying a candidate translocon from X. translucens. Notably, genomes from strains that lacked hrpF/popF/nolX instead encode another gene, called hpaT, adjacent to and co-regulated with the type III secretion system gene cluster. An insertional mutant in the X. translucens hpaT gene, which is the first gene of a two-gene operon, hpaT-hpaH, was non-pathogenic on barley and did not cause the HR or programmed cell death in non-host pepper similar to the hrcT mutant. The hpaT mutant phenotypes were partially complemented by either hpaT or the downstream gene, hpaH, which has been described as a facilitator of translocation in Xanthomonas oryzae. Interestingly, the hpaT mutant was also complemented

  19. Advice from working women with retired partners.

    Science.gov (United States)

    Cooley, Eileen L; Adorno, Gail

    2016-01-01

    in the 21st century, as more women are employed full-time and couples increasingly share egalitarian values, more women continue employment after their partners have voluntarily retired. However, we know very little about the experiences of this growing population of women. We asked working women with retired partners to share their advice for other women who may face this developmental transition. Open-ended responses from 97 women were analyzed to identify pertinent issues and themes. Four primary content areas were identified: time management, division of household labor, financial planning, and communication. Communication between partners was both a topic of concern as well as the solution suggested to resolve conflicts or differences that may arise when women live with a retired partner. It is expected that future changes in the workforce and improvements in the gender balance within relationships will continue to impact experiences for working women with retired partners.

  20. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    Science.gov (United States)

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

    Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for

  2. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

    DEFF Research Database (Denmark)

    Berquist, Brian R; Singh, Dharmendra Kumar; Fan, Jinshui

    2010-01-01

    XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R...... mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S...

  3. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

    DEFF Research Database (Denmark)

    Skjøt, Rikke L. V.; Oettinger, Thomas; Rosenkrands, Ida

    2000-01-01

    . The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P, B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998......), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

  4. Climate Ready Estuaries Partner Projects Map

    Science.gov (United States)

    CRE partners with the National Estuary Program to develop climate change projects in coastal U.S. areas, such as bays and harbors; to develop adaptation action plans, identify climate impacts and indicators, and more. This map shows project locations.

  5. How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

    Science.gov (United States)

    Zhan, Xianquan; Yang, Haiyan; Peng, Fang; Li, Jianglin; Mu, Yun; Long, Ying; Cheng, Tingting; Huang, Yuda; Li, Zhao; Lu, Miaolong; Li, Na; Li, Maoyu; Liu, Jianping; Jungblut, Peter R

    2018-04-01

    Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

    2015-11-06

    Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

  7. DichroMatch at the protein circular dichroism data bank (DM@PCDDB): A web-based tool for identifying protein nearest neighbors using circular dichroism spectroscopy.

    Science.gov (United States)

    Whitmore, Lee; Mavridis, Lazaros; Wallace, B A; Janes, Robert W

    2018-01-01

    Circular dichroism spectroscopy is a well-used, but simple method in structural biology for providing information on the secondary structure and folds of proteins. DichroMatch (DM@PCDDB) is an online tool that is newly available in the Protein Circular Dichroism Data Bank (PCDDB), which takes advantage of the wealth of spectral and metadata deposited therein, to enable identification of spectral nearest neighbors of a query protein based on four different methods of spectral matching. DM@PCDDB can potentially provide novel information about structural relationships between proteins and can be used in comparison studies of protein homologs and orthologs. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  8. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

    DEFF Research Database (Denmark)

    Skjøt, R L; Oettinger, T; Rosenkrands, I

    2000-01-01

    Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate....... The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998......), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

  9. Adaptive Evolution of Signaling Partners

    Science.gov (United States)

    Urano, Daisuke; Dong, Taoran; Bennetzen, Jeffrey L.; Jones, Alan M.

    2015-01-01

    Proteins that interact coevolve their structures. When mutation disrupts the interaction, compensation by the partner occurs to restore interaction otherwise counterselection occurs. We show in this study how a destabilizing mutation in one protein is compensated by a stabilizing mutation in its protein partner and their coevolving path. The pathway in this case and likely a general principle of coevolution is that the compensatory change must tolerate both the original and derived structures with equivalence in function and activity. Evolution of the structure of signaling elements in a network is constrained by specific protein pair interactions, by requisite conformational changes, and by catalytic activity. The heterotrimeric G protein-coupled signaling is a paragon of this protein interaction/function complexity and our deep understanding of this pathway in diverse organisms lends itself to evolutionary study. Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTP hydrolysis rate of the Gα subunit of the heterotrimeric G protein complex. An important RGS-contact site is a hydroxyl-bearing residue on the switch I region of Gα subunits in animals and most plants, such as Arabidopsis. The exception is the grasses (e.g., rice, maize, sugarcane, millets); these plants have Gα subunits that replaced the critical hydroxyl-bearing threonine with a destabilizing asparagine shown to disrupt interaction between Arabidopsis RGS protein (AtRGS1) and the grass Gα subunit. With one known exception (Setaria italica), grasses do not encode RGS genes. One parsimonious deduction is that the RGS gene was lost in the ancestor to the grasses and then recently acquired horizontally in the lineage S. italica from a nongrass monocot. Like all investigated grasses, S. italica has the Gα subunit with the destabilizing asparagine residue in the protein interface but, unlike other known grass genomes, still encodes an expressed RGS gene, SiRGS1. SiRGS1

  10. Proteomic Profiling of a Primary CD4+ T Cell Model of HIV-1 Latency Identifies Proteins Whose Differential Expression Correlates with Reactivation of Latent HIV-1.

    Science.gov (United States)

    Saha, Jamaluddin Md; Liu, Hongbing; Hu, Pei-Wen; Nikolai, Bryan C; Wu, Hulin; Miao, Hongyu; Rice, Andrew P

    2018-01-01

    The latent HIV-1 reservoir of memory CD4 + T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4 + T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.

  11. BLAST screening of chlamydial genomes to identify signature proteins that are unique for the Chlamydiales, Chlamydiaceae, Chlamydophila and Chlamydia groups of species

    Directory of Open Access Journals (Sweden)

    Gupta Radhey S

    2006-01-01

    Full Text Available Abstract Background Chlamydiae species are of much importance from a clinical viewpoint. Their diversity both in terms of their numbers as well as clinical involvement are presently believed to be significantly underestimated. The obligate intracellular nature of chlamydiae has also limited their genetic and biochemical studies. Thus, it is of importance to develop additional means for their identification and characterization. Results We have carried out analyses of available chlamydiae genomes to identify sets of unique proteins that are either specific for all Chlamydiales genomes, or different Chlamydiaceae family members, or members of the Chlamydia and Chlamydophila genera, or those unique to Protochlamydia amoebophila, but which are not found in any other bacteria. In total, 59 Chlamydiales-specific proteins, 79 Chlamydiaceae-specific proteins, 20 proteins each that are specific for both Chlamydia and Chlamydophila and 445 ORFs that are Protochlamydia-specific were identified. Additionally, 33 cases of possible gene loss or lateral gene transfer were also detected. Conclusion The identified chlamydiae-lineage specific proteins, many of which are highly conserved, provide novel biomarkers that should prove of much value in the diagnosis of these bacteria and in exploration of their prevalence and diversity. These conserved protein sequences (CPSs also provide novel therapeutic targets for drugs that are specific for these bacteria. Lastly, functional studies on these chlamydiae or chlamydiae subgroup-specific proteins should lead to important insights into lineage-specific adaptations with regards to development, infectivity and pathogenicity.

  12. Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

    Science.gov (United States)

    Wei, Wei; Luo, Weijia; Wu, Feilin; Peng, Xuehui; Zhang, Yao; Zhang, Manli; Zhao, Yan; Su, Na; Qi, YingZi; Chen, Lingsheng; Zhang, Yangjun; Wen, Bo; He, Fuchu; Xu, Ping

    2016-11-04

    Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

  13. Partner notification of sexually transmitted diseases: practices and preferences.

    Science.gov (United States)

    Gursahaney, Priya R; Jeong, Kwonho; Dixon, Bruce W; Wiesenfeld, Harold C

    2011-09-01

    Timely notification and treatment of sex partners exposed to a sexually transmitted disease (STD) is essential to reduce reinfection and transmission. Our objectives were to determine factors associated with patient-initiated notification of sex partners and preferences regarding standard partner referral versus expedited partner therapy (EPT). Participants diagnosed with gonorrhea, chlamydia, trichomoniasis, or nongonococcal urethritis within the previous year were administered a baseline survey asking about demographics, sexual history, and partner treatment preferences (standard partner referral vs. EPT). They identified up to 4 sex partners within the past 2 months, and answered questions on relationship characteristics, quality, and notification self-efficacy. At follow-up, participants with a current STD were asked whether they notified their partners. Generalized estimating equations were used to evaluate the associations between predictor variables and partner notification. Of the 201 subjects enrolled, 157 had a current STD diagnosis, and 289 sex partners were identified. The rate of successful partner notification was 77.3% (157/203 sex partners). Partner notification was increased if the subject had a long-term relationship with a sex partner (odds ratio: 3.07; 95% confidence interval: 1.43, 6.58), considered the partner to be a main partner (odds ratio: 2.53; 95% confidence interval: 1.43, 6.58), or had increased notification self-efficacy. Overall, participants did not prefer EPT over standard referral; however, females, those with higher education levels, and those with a prior STD preferred EPT. Patient-initiated partner referral is more successful in patients with increased self-efficacy who have stronger interpersonal relationships with their sex partners.

  14. Gathering and Using Information for the Selection of Trading Partners

    NARCIS (Netherlands)

    Nijssen, E.J.; Douglas, S.P.; Calis, G.

    1999-01-01

    Examines the nature of the search process used by international firms in identifying trading partners in emerging markets, and to what extent systematic information collection on potential partners is likely to enhance the choice of satisfactory partners. The results, based on 46 Dutch companies,

  15. Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

    Science.gov (United States)

    Canchignia, Hayron; Altimira, Fabiola; Montes, Christian; Sánchez, Evelyn; Tapia, Eduardo; Miccono, María; Espinoza, Daniel; Aguirre, Carlos; Seeger, Michael; Prieto, Humberto

    2017-03-17

    The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

  16. Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

    Directory of Open Access Journals (Sweden)

    Anantha Koteswararao Kanugula

    Full Text Available Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.

  17. New Partners in Regulation of Gene Expression: The Enhancer of Trithorax and Polycomb Corto Interacts with Methylated Ribosomal Protein L12 Via Its Chromodomain

    Science.gov (United States)

    Coléno-Costes, Anne; Jang, Suk Min; de Vanssay, Augustin; Rougeot, Julien; Bouceba, Tahar; Randsholt, Neel B.; Gibert, Jean-Michel; Le Crom, Stéphane; Mouchel-Vielh, Emmanuèle

    2012-01-01

    Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators. PMID:23071455

  18. Partner's influences and other correlates of prenatal alcohol use.

    Science.gov (United States)

    van der Wulp, Nickie Y; Hoving, Ciska; de Vries, Hein

    2015-04-01

    To investigate the influence of partners on alcohol consumption in pregnant women within the context of other factors. A Dutch nationwide online cross-sectional study among 158 pregnant women and their partners was conducted. To identify correlates of prenatal alcohol use, including perceived and reported partner norm (i.e. partner's belief regarding acceptability of prenatal alcohol use), partner modeling (i.e. partner's alcohol use during the woman's pregnancy) and partner support (i.e. partner's help in abstaining from alcohol during pregnancy), independent sample T-tests and Chi square tests were conducted. Correlation analyses tested the relationship between perceived and reported partner influence. Multivariate logistic hierarchical regression analyses tested the independent impact of partner's perceived and reported influence next to other correlates from the I-Change Model. Pregnant women who consumed alcohol perceived a weaker partner norm (p alcohol use and a weaker partner norm were more likely to use alcohol (R(2) = 0.42). This study demonstrated that perceived partner norm was the most critical of the constructs of perceived and reported partner influences in explaining prenatal alcohol use.

  19. Differential proteomic analysis to identify proteins associated with quality traits of frozen mud shrimp (Solenocera melantho) using an iTRAQ-based strategy.

    Science.gov (United States)

    Shi, Jing; Zhang, Longteng; Lei, Yutian; Shen, Huixing; Yu, Xunpei; Luo, Yongkang

    2018-06-15

    An iTRAQ-based strategy was applied to investigate proteome changes in mud shrimp during long-term frozen storage under different conditions. A total of 226 proteins was identified as differential abundance proteins (DAPs) in mud shrimp from two frozen treatment groups (-20 °C and -40 °C) compared with the fresh control group. The proteome changes in mud shrimp muscle stored under -20 °C was much greater than that under -40 °C. Correlation analysis between DAPs and quality traits of mud shrimp muscle showed that 12 proteins were correlated closely with color (L ∗ , a ∗ , and b ∗ value) and texture (hardness, elasticity, and chewiness). Bioinformatic analysis revealed that most of these proteins were involved in protein structure, metabolic enzymes, and protein turnover. Among them, several proteins might be potential protein markers for color, and some proteins are good candidate predictors for textural properties of mud shrimp muscle. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

    International Nuclear Information System (INIS)

    Chen, Liang; Chen, Huiqing; Yao, Chun; Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin; Chen, Keping

    2015-01-01

    Highlights: • On the cellular level, we identified IC 50 of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC 50 ) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress

  1. The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Liang; Chen, Huiqing [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Yao, Chun [Department of Stomatology, Zhenjiang First People’s Hospital, Zhenjiang, Jiangsu 212013 (China); Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Chen, Keping, E-mail: kpchen@ujs.edu.cn [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China)

    2015-04-09

    Highlights: • On the cellular level, we identified IC{sub 50} of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC{sub 50}) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress.

  2. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    Science.gov (United States)

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The association of 83 plasma proteins with CHD mortality, BMI, HDL-, and total-cholesterol in men: applying multivariate statistics to identify proteins with prognostic value and biological relevance.

    Science.gov (United States)

    Heidema, A Geert; Thissen, Uwe; Boer, Jolanda M A; Bouwman, Freek G; Feskens, Edith J M; Mariman, Edwin C M

    2009-06-01

    In this study, we applied the multivariate statistical tool Partial Least Squares (PLS) to analyze the relative importance of 83 plasma proteins in relation to coronary heart disease (CHD) mortality and the intermediate end points body mass index, HDL-cholesterol and total cholesterol. From a Dutch monitoring project for cardiovascular disease risk factors, men who died of CHD between initial participation (1987-1991) and end of follow-up (January 1, 2000) (N = 44) and matched controls (N = 44) were selected. Baseline plasma concentrations of proteins were measured by a multiplex immunoassay. With the use of PLS, we identified 15 proteins with prognostic value for CHD mortality and sets of proteins associated with the intermediate end points. Subsequently, sets of proteins and intermediate end points were analyzed together by Principal Components Analysis, indicating that proteins involved in inflammation explained most of the variance, followed by proteins involved in metabolism and proteins associated with total-C. This study is one of the first in which the association of a large number of plasma proteins with CHD mortality and intermediate end points is investigated by applying multivariate statistics, providing insight in the relationships among proteins, intermediate end points and CHD mortality, and a set of proteins with prognostic value.

  4. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  5. Collateral Intimate Partner Homicide

    Directory of Open Access Journals (Sweden)

    Emily Meyer

    2013-04-01

    Full Text Available Collateral intimate partner homicide (CIPH is an underinvestigated genre of intimate partner violence (IPV where an individual(s connected to the IPV victim is murdered. We conducted a content analysis of a statewide database of CIPH newspaper articles (1990-2007. Out of 111 collateral murder victims, there were 84 IPV female focal victims and 84 male perpetrators. The most frequently reported CIPH decedent was the focal victim’s new partner (30%; 45% of focal victims were themselves killed. News reports framed CIPH as the unexpected result of interpersonal conflict, despite evidence of a systematic pattern of coercion and violence that capitulated in murder.

  6. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  7. BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

    International Nuclear Information System (INIS)

    Markley, John L.; Ulrich, Eldon L.; Berman, Helen M.; Henrick, Kim; Nakamura, Haruki; Akutsu, Hideo

    2008-01-01

    We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems

  8. A Virtual Research Partner

    National Research Council Canada - National Science Library

    Cowie, Jim; Guerrero, Felicia

    2006-01-01

    .... The goal was to investigate the feasibility of creating a software agent that would be able to interact with researchers and provide them with support at a level equivalent to a human research partner...

  9. Green Power Partner List

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership is a voluntary program designed to reduce the environmental impact of electricity generation by promoting renewable energy. There are thousands of Green Power Partners, all listed on this page.

  10. CHP Partnership Partners

    Science.gov (United States)

    Partners of EPA's Combined Heat and Power Partnership include federal, state, and local government agencies and private organizations such as energy users, energy service companies, CHP project developers and consultants, and equipment manufacturers.

  11. Multiple sex partner

    African Journals Online (AJOL)

    User

    intercourse, about 60% reported having a single sexual partner and 40% reported having multiple ... masturbation, start having sex at a younger age, have sex with married people and/or .... sex were considered unacceptable by 89 vs.

  12. Profiling of Parkin-binding partners using tandem affinity purification.

    Directory of Open Access Journals (Sweden)

    Alessandra Zanon

    Full Text Available Parkinson's disease (PD is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2 are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells, we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

  13. Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

    Science.gov (United States)

    Blankenburg, Hagen; Doncheva, Nadezhda T.; Schwienbacher, Christine; Serafin, Alice; Alexa, Adrian; Weichenberger, Christian X.; Albrecht, Mario; Klein, Christine; Hicks, Andrew A.; Pramstaller, Peter P.

    2013-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function. PMID:24244333

  14. Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease: Brain protein O-GlcNAcylation in Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sheng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Yang, Feng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Petyuk, Vladislav A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Shukla, Anil K. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gritsenko, Marina A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Rodland, Karin D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Qian, Wei-Jun [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gong, Cheng-Xin [New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York USA; Liu, Tao [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA

    2017-07-28

    Protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer’s disease. Herein we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in post-mortem human brains with and without Alzheimer’s using isobaric tandem mass tags labeling, chemoenzymatic photocleavage enrichment and liquid chromatography coupled to mass spectrometry. A total of 1,850 O-GlcNAc peptides covering 1,094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. 128 O-GlcNAc peptides covering 78 proteins were altered significantly in Alzheimer’s brain as compared to controls (q<0.05). Moreover, alteration of the O-GlcNAc peptide abundance could be attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic Alzheimer’s disease.

  15. A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Friedmann Theodore

    2009-08-01

    Full Text Available Abstract Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1, relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

  16. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    International Nuclear Information System (INIS)

    Rossier, Olivier; Giannone, Grégory

    2016-01-01

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  17. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    Energy Technology Data Exchange (ETDEWEB)

    Rossier, Olivier; Giannone, Grégory [Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France); CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France)

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  18. The lonely female partner

    DEFF Research Database (Denmark)

    Bruun, Poul; Pedersen, Birthe D; Osther, Palle J

    2011-01-01

    The aim of this qualitative study was to investigate the experiences of female partners to men with prostate cancer. The women found the capacity to manage their lives through mutual love in the family and through their faith.......The aim of this qualitative study was to investigate the experiences of female partners to men with prostate cancer. The women found the capacity to manage their lives through mutual love in the family and through their faith....

  19. Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

    Science.gov (United States)

    Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

    2018-05-01

    Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

  20. Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

    Science.gov (United States)

    Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

    2014-09-01

    Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

  1. Genome-wide analysis of gene expression and protein secretion of Babesia canis during virulent infection identifies potential pathogenicity factors.

    Science.gov (United States)

    Eichenberger, Ramon M; Ramakrishnan, Chandra; Russo, Giancarlo; Deplazes, Peter; Hehl, Adrian B

    2017-06-13

    Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite's exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.

  2. Identification and characterization of Iporin as a novel interaction partner for rab1

    Directory of Open Access Journals (Sweden)

    Konczal Magdalena

    2005-03-01

    Full Text Available Abstract Background The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. Results Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD. Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. Conclusion Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.

  3. In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.

    Directory of Open Access Journals (Sweden)

    Aihua Zheng

    2010-10-01

    Full Text Available Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. .

  5. El personal sanitario no percibe la violencia doméstica como un problema de salud Intimate partner violence is not identified as a health problem by health care workers

    Directory of Open Access Journals (Sweden)

    Blanca Coll-Vinent

    2008-02-01

    Full Text Available Objetivo: Evaluar la opinión del personal sanitario de nuestro hospital frente al fenómeno de la violencia doméstica. Métodos: Estudio descriptivo de prevalencia, utilizando un cuestionario diseñado ad hoc. Se preguntó a 321 profesionales, seleccionados aleatoriamente, acerca de la violencia doméstica y su relación con la salud. Resultados: Respondieron 287 profesionales. La mayoría de encuestados (87,1% consideró que era un problema importante, pero no lo consideraban un problema de salud. El 60% consideró que podía tener un papel relevante en la detección de esta situación. En general, valoraron como muy baja su preparación para atender a este tipo de pacientes. El personal de enfermería era más sensible al problema y se consideraba más preparado que el personal médico. Conclusiones: Los profesionales de la salud están sensibilizados frente al problema de la violencia de género, pero no lo consideran un problema de salud y aducen falta de preparación para abordar este tema.Objective: To determine attitudes, opinions and knowledge of domestic violence among medical and nursing staff. Methods: We performed a descriptive study of prevalence using an ad hoc questionnaire. A random sample of 321 health professionals were asked about their opinions and knowledge of domestic violence and its relationship with health. Results: A total of 287 health professions completed the questionnaire. Most (87.1% considered that intimate partner violence was an important problem, but not a health issue. Sixty percent of the health professionals believed that they could play an important role in detecting patients in this situation. Knowledge about the management of this problem was low. Nursing professionals were more sensitive to this issue and better prepared than medical staff. Conclusions: Healthcare personnel are sensitive to the problem of intimate partner violence but do not consider this issue to be a health problem. Education about

  6. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

    DEFF Research Database (Denmark)

    Skjøt, R L; Oettinger, T; Rosenkrands, I

    2000-01-01

    Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtra...

  7. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

    Science.gov (United States)

    Rossier, Olivier; Giannone, Grégory

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

  8. Bioinformatical Analysis of Organ-Related (Heart, Brain, Liver, and Kidney and Serum Proteomic Data to Identify Protein Regulation Patterns and Potential Sepsis Biomarkers

    Directory of Open Access Journals (Sweden)

    Andreas Hohn

    2018-01-01

    Full Text Available During the last years, proteomic studies have revealed several interesting findings in experimental sepsis models and septic patients. However, most studies investigated protein alterations only in single organs or in whole blood. To identify possible sepsis biomarkers and to evaluate the relationship between protein alteration in sepsis affected organs and blood, proteomics data from the heart, brain, liver, kidney, and serum were analysed. Using functional network analyses in combination with hierarchical cluster analysis, we found that protein regulation patterns in organ tissues as well as in serum are highly dynamic. In the tissue proteome, the main functions and pathways affected were the oxidoreductive activity, cell energy generation, or metabolism, whereas in the serum proteome, functions were associated with lipoproteins metabolism and, to a minor extent, with coagulation, inflammatory response, and organ regeneration. Proteins from network analyses of organ tissue did not correlate with statistically significantly regulated serum proteins or with predicted proteins of serum functions. In this study, the combination of proteomic network analyses with cluster analyses is introduced as an approach to deal with high-throughput proteomics data to evaluate the dynamics of protein regulation during sepsis.

  9. Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein

    International Nuclear Information System (INIS)

    Shipp, M.A.; Richardson, N.E.; Sayre, P.H.; Brown, N.R.; Masteller, E.L.; Clayton, L.K.; Ritz, J.; Reinherz, E.L.

    1988-01-01

    Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate the primary structure of CALLA, the authors purified the protein to homogeneity, obtained the NH 2 -terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line λgt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NM 2 -terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA + cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types

  10. Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

    NARCIS (Netherlands)

    Lu, J.; Boeren, J.A.; Vries, de S.C.; Valenberg, van H.J.F.; Vervoort, J.J.M.; Hettinga, K.A.

    2011-01-01

    Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as

  11. Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics*

    Science.gov (United States)

    Subota, Ines; Julkowska, Daria; Vincensini, Laetitia; Reeg, Nele; Buisson, Johanna; Blisnick, Thierry; Huet, Diego; Perrot, Sylvie; Santi-Rocca, Julien; Duchateau, Magalie; Hourdel, Véronique; Rousselle, Jean-Claude; Cayet, Nadège; Namane, Abdelkader; Chamot-Rooke, Julia; Bastin, Philippe

    2014-01-01

    Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. PMID:24741115

  12. Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

    Science.gov (United States)

    Subota, Ines; Julkowska, Daria; Vincensini, Laetitia; Reeg, Nele; Buisson, Johanna; Blisnick, Thierry; Huet, Diego; Perrot, Sylvie; Santi-Rocca, Julien; Duchateau, Magalie; Hourdel, Véronique; Rousselle, Jean-Claude; Cayet, Nadège; Namane, Abdelkader; Chamot-Rooke, Julia; Bastin, Philippe

    2014-07-01

    Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. © 2014 by The

  13. Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project.

    Science.gov (United States)

    Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Kim, Kwang-Youl; Kwon, Kyung-Hoon; Yoo, Jong Shin; Omenn, Gilbert S; Baker, Mark S; Hancock, William S; Paik, Young-Ki

    2015-12-04

    Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library

  14. Genital Herpes in Marital Partners

    Directory of Open Access Journals (Sweden)

    Mary Jacob

    1988-01-01

    Full Text Available During 1983-86, 225 patients were clinically diagnosed to have genital herpes (GH at our clinic. Of these, 90 men and 55 women were currently married. All the spouses were screened clinically and through standardized techniques for isolation and typing of herpes simplex virus, serological testing and Papanicolaou smear. There were 90 couples in whom at least one spouse had GH and in 38 (42% couples both partners had GH. Clinically, 49% of wives and 75% of husbands of GH patients were diagnosed to have the disease. The spouses of recurrent GH patients had a higher frequency of the disease than spouses of primary GH patients. Among spouses who were clinically asymptomatic, 40% had high serological titres suggestive of GH. Wives generally experienced more severe symptoms, especially pain in the lesions. Majority of lesions in both the partners were vesicles and ulcers. Prodromata were more among recurrent GH patients in both the partners. The frequency of recurrences wasalso similar in spouses. Seventy percent of wives and 40% of husbands could not identify any precipitating factor. Intercourse, physical stress and rich food were cited as possible factors in the remaining. All the wives had acquired the diseases through their husbands who were promiscuous. Fifty percent of husbands had been infected before marriage. Given the fact that asymptomatic carriers exist, it is better to consider all marital partners of GH as infected. Repeated and long-term follow, - up examination, particularly of wives of GH patients is therefore essential as an important socio-preventive aspect of this disease.

  15. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck

    2012-01-01

    , is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC...... the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate...... regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated...

  16. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

    Science.gov (United States)

    Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

    2018-01-01

    Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  17. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase

    Czech Academy of Sciences Publication Activity Database

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, L.-Y.; Gelvin, S.B.; Sýkorová, Eva

    2015-01-01

    Roč. 6, NOV2015 (2015) ISSN 1664-462X R&D Projects: GA ČR GA13-06943S; GA MŠk(CZ) ED1.1.00/02.0068 Grant - others:GA MŠk(CZ) LH10352 Institutional support: RVO:68081707 ; RVO:61389030 Keywords : telomerase * nuclear poly(A)-binding protein * telobox Subject RIV: BO - Biophysics; EF - Botanics (UEB-Q) Impact factor: 4.495, year: 2015

  18. A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

    Science.gov (United States)

    2015-07-27

    total protein in each sample was quantified by Bradford assay (Bio-Rad). RAW264.7 cell lysate preparations were boiled for 10 min with NuPAGE LDS Sample...RPMA assays , cells were harvested, washed with PBS, and then lysed in a mixture of T-PER Reagent (Thermo Scientific) and 2X Tris-Glycine SDS sample... assay (RIPA) buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche), and phosphatase inhibitors (Roche). The amount of

  19. Identifying Potential Protein Targets for Toluene Using a Molecular Similarity Search, in Silico Docking and in Vitro Validation

    Science.gov (United States)

    2015-01-01

    performed under standard conditions. Ana- lysis of purified hemoglobin using SDS and native polyacryl - amide gel electrophoresis (PAGE) indicated that the...search of T3DB. They represent several families of proteins (calcium-transporting ATPases, sodium/ potassium -transporting ATPase, cytochrome P450...REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1

  20. Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpio L.

    Science.gov (United States)

    Gotesman, M; Soliman, H; El-Matbouli, M

    2014-01-01

    Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study. PMID:23347276

  1. Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response.

    Science.gov (United States)

    Long, J; Fang, W Y; Chang, L; Gao, W H; Shen, Y; Jia, M Y; Zhang, Y X; Wang, Y; Dou, H B; Zhang, W J; Zhu, J; Liang, A B; Li, J M; Hu, Jiong

    2017-12-01

    Resistance to cytotoxic chemotherapy drugs remains as the major cause of treatment failure in acute myeloid leukemia. Histone deacetylases (HDAC) are important regulators to maintain chromatin structure and control DNA damage; nevertheless, how each HDAC regulates genome stability remains unclear, especially under genome stress conditions. Here, we identified a mechanism by which HDAC3 regulates DNA damage repair and mediates resistance to chemotherapy drugs. In addition to inducing DNA damage, chemotherapy drugs trigger upregulation of HDAC3 expression in leukemia cells. Using genetic and pharmacological approaches, we show that HDAC3 contributes to chemotherapy resistance by regulating the activation of AKT, a well-documented factor in drug resistance development. HDAC3 binds to AKT and deacetylates it at the site Lys20, thereby promoting the phosphorylation of AKT. Chemotherapy drug exposure enhances the interaction between HDAC3 and AKT, resulting in decrease in AKT acetylation and increase in AKT phosphorylation. Whereas HDAC3 depletion or inhibition abrogates these responses and meanwhile sensitizes leukemia cells to chemotoxicity-induced apoptosis. Importantly, in vivo HDAC3 suppression reduces leukemia progression and sensitizes MLL-AF9 + leukemia to chemotherapy. Our findings suggest that combination therapy with HDAC3 inhibitor and genotoxic agents may constitute a successful strategy for overcoming chemotherapy resistance.

  2. Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

    Science.gov (United States)

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

    Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

  3. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins

    NARCIS (Netherlands)

    Macaulay, Iain C.; Tijssen, Marloes R.; Thijssen-Timmer, Daphne C.; Gusnanto, Arief; Steward, Michael; Burns, Philippa; Langford, Cordelia F.; Ellis, Peter D.; Dudbridge, Frank; Zwaginga, Jaap-Jan; Watkins, Nicholas A.; van der Schoot, C. Ellen; Ouwehand, Willem H.

    2007-01-01

    To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of

  4. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    Chunyan Mo

    2018-03-01

    Full Text Available Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL protein and CBL-interacting protein kinase (CIPK is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL and 26 CIPK (MeCIPK genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR, and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  5. Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach

    Directory of Open Access Journals (Sweden)

    Choi Young J

    2006-08-01

    Full Text Available Abstract Background In the interest of generating large amounts of recombinant protein, inducible systems have been studied to maximize both the growth of the culture and the production of foreign proteins. Even though thermo-inducible systems were developed in the late 1970's, the number of studies that focus on strategies for the implementation at bioreactor scale is limited. In this work, the bacteriophage lambda PL promoter is once again investigated as an inducible element but for the production of green fluorescent protein (GFP. Culture temperature, induction point, induction duration and number of inductions were considered as factors to maximize GFP production in a 20-L bioreactor. Results It was found that cultures carried out at 37°C resulted in a growth-associated production of GFP without the need of an induction at 42°C. Specific production was similar to what was achieved when separating the growth and production phases. Shake flask cultures were used to screen for desirable operating conditions. It was found that multiple inductions increased the production of GFP. Induction decreased the growth rate and substrate yield coefficients; therefore, two time domains (before and after induction having different kinetic parameters were created to fit a model to the data collected. Conclusion Based on two batch runs and the simulation of culture dynamics, a pre-defined feeding and induction strategy was developed to increase the volumetric yield of a temperature regulated expression system and was successfully implemented in a 20-L bioreactor. An overall cell density of 5.95 g DW l-1 was achieved without detriment to the cell specific production of GFP; however, the production of GFP was underestimated in the simulations due to a significant contribution of non-growth associated product formation under limiting nutrient conditions.

  6. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    Directory of Open Access Journals (Sweden)

    Rudo L Simeon

    Full Text Available Genetic suppressor elements (GSEs are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

  7. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins.

    Science.gov (United States)

    Hendrikx, Lotte H; Oztürk, Kemal; de Rond, Lia G H; Veenhoven, Reinier H; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-02-04

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore protection against pertussis may depend largely on long-term B- and T-cell immunities. We investigated long-term pertussis-specific memory B-cell responses in children who were primed at infant age with the Dutch wP-vaccine (ISRCTN65428640). Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin, pertactin and tetanus. In addition, plasma IgG levels directed to the same antigens were measured by a fluorescent bead-based multiplex immunoassay. Two and 3 years after wP priming as well as 2 and 5 years after the aP booster at the age of 4, low plasma IgG levels to the pertussis proteins were found. At the same time, however pertussis protein-specific memory B-cells could be detected and their number increased with age. The number of tetanus-specific memory B-cells was similar in all age groups, whereas IgG-tetanus levels were high 2 years after tetanus booster compared to pre- and 5 years post-booster levels. This study shows the presence of long-term pertussis protein-specific memory B-cells in children despite waning antibody levels after vaccination, which suggests that memory B-cells in addition to antibodies may contribute to protection against pertussis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

    Czech Academy of Sciences Publication Activity Database

    Hafidh, Said; Potěšil, D.; Fíla, Jan; Čapková, Věra; Zdráhal, Z.; Honys, David

    2016-01-01

    Roč. 17, MAY 3 (2016), č. článku 81. ISSN 1465-6906 R&D Projects: GA ČR GA15-22720S; GA ČR(CZ) GA14-32292S; GA ČR(CZ) GA15-16050S; GA MŠk(CZ) LD14109; GA MŠk(CZ) LM2015043; GA MŠk(CZ) ED1.1.00/02.0068; GA MŠk(CZ) LQ1601 Institutional support: RVO:61389030 Keywords : Protein secretion * Pollen tube guidance * Cell-cell signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 11.313, year: 2015

  9. HomPPI: a class of sequence homology based protein-protein interface prediction methods

    Directory of Open Access Journals (Sweden)

    Dobbs Drena

    2011-06-01

    Full Text Available Abstract Background Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate. Results We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i NPS-HomPPI (Non partner-specific HomPPI, which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii PS-HomPPI (Partner-specific HomPPI, which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of

  10. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    Science.gov (United States)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  11. Single-molecule spectroscopy of LHCSR1 protein dynamics identifies two distinct states responsible for multi-timescale photosynthetic photoprotection

    Science.gov (United States)

    Kondo, Toru; Pinnola, Alberta; Chen, Wei Jia; Dall'Osto, Luca; Bassi, Roberto; Schlau-Cohen, Gabriela S.

    2017-08-01

    In oxygenic photosynthesis, light harvesting is regulated to safely dissipate excess energy and prevent the formation of harmful photoproducts. Regulation is known to be necessary for fitness, but the molecular mechanisms are not understood. One challenge has been that ensemble experiments average over active and dissipative behaviours, preventing identification of distinct states. Here, we use single-molecule spectroscopy to uncover the photoprotective states and dynamics of the light-harvesting complex stress-related 1 (LHCSR1) protein, which is responsible for dissipation in green algae and moss. We discover the existence of two dissipative states. We find that one of these states is activated by pH and the other by carotenoid composition, and that distinct protein dynamics regulate these states. Together, these two states enable the organism to respond to two types of intermittency in solar intensity—step changes (clouds and shadows) and ramp changes (sunrise), respectively. Our findings reveal key control mechanisms underlying photoprotective dissipation, with implications for increasing biomass yields and developing robust solar energy devices.

  12. Purification and subunit structure of a putative K sup + -channel protein identified by its binding properties for dendrotoxin I

    Energy Technology Data Exchange (ETDEWEB)

    Rehm, H.; Lazdunski, M. (Centre National de la Recherche Scientifique, Nice (France))

    1988-07-01

    The binding protein for the K{sup +}-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K{sub d}, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO{sub 4}/polyacrylamide gel revealed three bands of M{sub r} 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for {sup 125}I-labeled mast cell degranulating peptide, another putative K{sup +}-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.

  13. Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I

    International Nuclear Information System (INIS)

    Rehm, H.; Lazdunski, M.

    1988-01-01

    The binding protein for the K + -channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K d , 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO 4 /polyacrylamide gel revealed three bands of M r 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for 125 I-labeled mast cell degranulating peptide, another putative K + -channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol

  14. Design of multi-specificity in protein interfaces.

    Directory of Open Access Journals (Sweden)

    Elisabeth L Humphris

    2007-08-01

    Full Text Available Interactions in protein networks may place constraints on protein interface sequences to maintain correct and avoid unwanted interactions. Here we describe a "multi-constraint" protein design protocol to predict sequences optimized for multiple criteria, such as maintaining sets of interactions, and apply it to characterize the mechanism and extent to which 20 multi-specific proteins are constrained by binding to multiple partners. We find that multi-specific binding is accommodated by at least two distinct patterns. In the simplest case, all partners share key interactions, and sequences optimized for binding to either single or multiple partners recover only a subset of native amino acid residues as optimal. More interestingly, for signaling interfaces functioning as network "hubs," we identify a different, "multi-faceted" mode, where each binding partner prefers its own subset of wild-type residues within the promiscuous binding site. Here, integration of preferences across all partners results in sequences much more "native-like" than seen in optimization for any single binding partner alone, suggesting these interfaces are substantially optimized for multi-specificity. The two strategies make distinct predictions for interface evolution and design. Shared interfaces may be better small molecule targets, whereas multi-faceted interactions may be more "designable" for altered specificity patterns. The computational methodology presented here is generalizable for examining how naturally occurring protein sequences have been selected to satisfy a variety of positive and negative constraints, as well as for rationally designing proteins to have desired patterns of altered specificity.

  15. Na+/K+-ATPase α1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role in the barrier integrity.

    Directory of Open Access Journals (Sweden)

    Yue Yang

    2011-01-01

    Full Text Available The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+/K(+-ATPase α1 (ATP1A1 with protein kinase C eta (PKCη and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.The results presented here provide a novel method for

  16. Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Taddei Kevin

    2010-01-01

    Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

  17. Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Bose, Mahuya; Adams, Brian P; Whittal, Randy M; Bose, Himangshu S

    2008-02-01

    Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

  18. Intimate partner violence (IPV)

    DEFF Research Database (Denmark)

    Rasch, Vibeke; Van, Toan Ngo; Nguyen, Hanh Thi Thuy

    2018-01-01

    BACKGROUND: Intimate partner violence (IPV) is a global problem that affects one-third of all women. The present study aims to develop and determine the validity of a screening instrument for the detection of IPV in pregnant women in Tanzania and Vietnam and to determine the minimum number...

  19. The stress granule component G3BP is a novel interaction partner for the nuclear shuttle proteins of the nanovirus pea necrotic yellow dwarf virus and geminivirus abutilon mosaic virus.

    Science.gov (United States)

    Krapp, Susanna; Greiner, Eva; Amin, Bushra; Sonnewald, Uwe; Krenz, Björn

    2017-01-02

    Stress granules (SGs) are structures within cells that regulate gene expression during stress response, e.g. viral infection. In mammalian cells assembly of SGs is dependent on the Ras-GAP SH3-domain-binding protein (G3BP). The C-terminal domain of the viral nonstructural protein 3 (nsP3) of Semliki Forest virus (SFV) forms a complex with mammalian G3BP and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs. The binding domain of nsP3 to HsG3BP was mapped to two tandem 'FGDF' repeat motifs close to the C-terminus of the viral proteins. It was speculated that plant viruses employ a similar strategy to inhibit SG function. This study identifies an Arabidopsis thaliana NTF2-RRM domain-containing protein as a G3BP-like protein (AtG3BP), which localizes to plant SGs. Moreover, the nuclear shuttle protein (NSP) of the begomovirus abutilon mosaic virus (AbMV), which harbors a 'FVSF'-motif at its C-terminal end, interacts with the AtG3BP-like protein, as does the 'FNGSF'-motif containing NSP of pea necrotic yellow dwarf virus (PNYDV), a member of the Nanoviridae family. We therefore propose that SG formation upon stress is conserved between mammalian and plant cells and that plant viruses may follow a similar strategy to inhibit plant SG function as it has been shown for their mammalian counterparts. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    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.

  1. Predictability of Conversation Partners

    Science.gov (United States)

    Takaguchi, Taro; Nakamura, Mitsuhiro; Sato, Nobuo; Yano, Kazuo; Masuda, Naoki

    2011-08-01

    Recent developments in sensing technologies have enabled us to examine the nature of human social behavior in greater detail. By applying an information-theoretic method to the spatiotemporal data of cell-phone locations, [C. Song , ScienceSCIEAS0036-8075 327, 1018 (2010)] found that human mobility patterns are remarkably predictable. Inspired by their work, we address a similar predictability question in a different kind of human social activity: conversation events. The predictability in the sequence of one’s conversation partners is defined as the degree to which one’s next conversation partner can be predicted given the current partner. We quantify this predictability by using the mutual information. We examine the predictability of conversation events for each individual using the longitudinal data of face-to-face interactions collected from two company offices in Japan. Each subject wears a name tag equipped with an infrared sensor node, and conversation events are marked when signals are exchanged between sensor nodes in close proximity. We find that the conversation events are predictable to a certain extent; knowing the current partner decreases the uncertainty about the next partner by 28.4% on average. Much of the predictability is explained by long-tailed distributions of interevent intervals. However, a predictability also exists in the data, apart from the contribution of their long-tailed nature. In addition, an individual’s predictability is correlated with the position of the individual in the static social network derived from the data. Individuals confined in a community—in the sense of an abundance of surrounding triangles—tend to have low predictability, and those bridging different communities tend to have high predictability.

  2. Predictability of Conversation Partners

    Directory of Open Access Journals (Sweden)

    Taro Takaguchi

    2011-09-01

    Full Text Available Recent developments in sensing technologies have enabled us to examine the nature of human social behavior in greater detail. By applying an information-theoretic method to the spatiotemporal data of cell-phone locations, [C. Song et al., Science 327, 1018 (2010SCIEAS0036-8075] found that human mobility patterns are remarkably predictable. Inspired by their work, we address a similar predictability question in a different kind of human social activity: conversation events. The predictability in the sequence of one’s conversation partners is defined as the degree to which one’s next conversation partner can be predicted given the current partner. We quantify this predictability by using the mutual information. We examine the predictability of conversation events for each individual using the longitudinal data of face-to-face interactions collected from two company offices in Japan. Each subject wears a name tag equipped with an infrared sensor node, and conversation events are marked when signals are exchanged between sensor nodes in close proximity. We find that the conversation events are predictable to a certain extent; knowing the current partner decreases the uncertainty about the next partner by 28.4% on average. Much of the predictability is explained by long-tailed distributions of interevent intervals. However, a predictability also exists in the data, apart from the contribution of their long-tailed nature. In addition, an individual’s predictability is correlated with the position of the individual in the static social network derived from the data. Individuals confined in a community—in the sense of an abundance of surrounding triangles—tend to have low predictability, and those bridging different communities tend to have high predictability.

  3. Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

    2016-05-10

    Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

  4. CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

    Science.gov (United States)

    Hafsa, Noor E; Arndt, David; Wishart, David S

    2015-07-01

    The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Evaluation of two recombinant Leishmania proteins identified by an immunoproteomic approach as tools for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

    Science.gov (United States)

    Coelho, Eduardo Antonio Ferraz; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Garde, Esther; de Jesus Pereira, Nathália Cristina; Lopes, Eliane Gonçalves Paiva; Borges, Luiz Felipe Nunes Menezes; Duarte, Mariana Costa; Menezes-Souza, Daniel; de Magalhães-Soares, Danielle Ferreira; Chávez-Fumagalli, Miguel Angel; Soto, Manuel; Tavares, Carlos Alberto Pereira

    2016-01-15

    Serological diagnostic tests for canine and human leishmaniasis present problems related with their sensitivity and/or specificity. Recently, an immunoproteomic approach performed with Leishmania infantum proteins identified new parasite antigens. In the present study, the diagnostic properties of two of these proteins, cytochrome c oxidase and IgE-dependent histamine-releasing factor, were evaluated for the serodiagnosis of canine visceral (CVL) and human tegumentary (HTL) leishmaniasis. For the CVL diagnosis, sera samples from non-infected dogs living in an endemic or non-endemic area of leishmaniasis, sera from asymptomatic or symptomatic visceral leishmaniasis (VL) dogs, from Leish-Tec(®)-vaccinated dogs, and sera from animals experimentally infected by Trypanosoma cruzi or Ehrlichia canis were used. For the HTL diagnosis, sera from non-infected subjects living in an endemic area of leishmaniasis, sera from active cutaneous or mucosal leishmaniasis patients, as well as those from T. cruzi-infected patients were employed. ELISA assays using the recombinant proteins showed both sensitivity and specificity values of 100% for the serodiagnosis of both forms of disease, with high positive and negative predictive values, showing better diagnostic properties than the parasite recombinant A2 protein or a soluble Leishmania antigen extract. In this context, the two new recombinant proteins could be considered to be used in the serodiagnosis of CVL and HTL. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

    Science.gov (United States)

    Jaiswal, Dinesh Kumar; Mishra, Poonam; Subba, Pratigya; Rathi, Divya; Chakraborty, Subhra; Chakraborty, Niranjan

    2014-02-01

    Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.

  7. Proteome analysis identifies the Dpr protein of Streptococcus mutans as an important factor in the presence of early streptococcal colonizers of tooth surfaces.

    Directory of Open Access Journals (Sweden)

    Akihiro Yoshida

    Full Text Available Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE. After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci.

  8. Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

    Science.gov (United States)

    Yoshida, Akihiro; Niki, Mamiko; Yamamoto, Yuji; Yasunaga, Ai; Ansai, Toshihiro

    2015-01-01

    Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci. PMID:25816242

  9. Partner Choice in Raven (Corvus corax) Cooperation.

    Science.gov (United States)

    Asakawa-Haas, Kenji; Schiestl, Martina; Bugnyar, Thomas; Massen, Jorg J M

    2016-01-01

    Although social animals frequently make decisions about when or with whom to cooperate, little is known about the underlying mechanisms of partner choice. Most previous studies compared different dyads' performances, though did not allow an actual choice among partners. We tested eleven ravens, Corvus corax, in triads, giving them first the choice to cooperate with either a highly familiar or a rather unfamiliar partner and, second, with either a friend or a non-friend using a cooperative string-pulling task. In either test, the ravens had a second choice and could cooperate with the other partner, given that this one had not pulled the string in the meantime. We show that during the experiments, these partner ravens indeed learn to wait and inhibit pulling, respectively. Moreover, the results of these two experiments show that ravens' preferences for a specific cooperation partner are not based on familiarity. In contrast, the ravens did show a preference based on relationship quality, as they did choose to cooperate significantly more with friends than with non-friends and they were also more proficient when cooperating with a friend. In order to further identify the proximate mechanism of this preference, we designed an open-choice experiment for the whole group where all birds were free to cooperate on two separate apparatuses. This set-up allowed us to distinguish between preferences for close proximity and preferences to cooperate. The results revealed that friends preferred staying close to each other, but did not necessarily cooperate with one another, suggesting that tolerance of proximity and not relationship quality as a whole may be the driving force behind partner choice in raven cooperation. Consequently, we stress the importance of experiments that allow such titrations and, suggest that these results have important implications for the interpretations of cooperation studies that did not include open partner choice.

  10. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  11. An effective approach for annotation of protein families with low sequence similarity and conserved motifs: identifying GDSL hydrolases across the plant kingdom.

    Science.gov (United States)

    Vujaklija, Ivan; Bielen, Ana; Paradžik, Tina; Biđin, Siniša; Goldstein, Pavle; Vujaklija, Dušica

    2016-02-18

    The massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom. Motif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through

  12. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    Directory of Open Access Journals (Sweden)

    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  13. Identifying molecular effects of diet through systems biology: influence of herring diet on sterol metabolism and protein turnover in mice.

    Directory of Open Access Journals (Sweden)

    Intawat Nookaew

    Full Text Available BACKGROUND: Changes in lifestyle have resulted in an epidemic development of obesity-related diseases that challenge the healthcare systems worldwide. To develop strategies to tackle this problem the focus is on diet to prevent the development of obesity-associated diseases such as cardiovascular disease (CVD. This will require methods for linking nutrient intake with specific metabolic processes in different tissues. METHODOLOGY/PRINCIPAL FINDING: Low-density lipoprotein receptor-deficient (Ldlr -/- mice were fed a high fat/high sugar diet to mimic a westernized diet, being a major reason for development of obesity and atherosclerosis. The diets were supplemented with either beef or herring, and matched in macronutrient contents. Body composition, plasma lipids and aortic lesion areas were measured. Transcriptomes of metabolically important tissues, e.g. liver, muscle and adipose tissue were analyzed by an integrated approach with metabolic networks to directly map the metabolic effects of diet in these different tissues. Our analysis revealed a reduction in sterol metabolism and protein turnover at the transcriptional level in herring-fed mice. CONCLUSION: This study shows that an integrated analysis of transcriptome data using metabolic networks resulted in the identification of signature pathways. This could not have been achieved using standard clustering methods. In particular, this systems biology analysis could enrich the information content of biomedical or nutritional data where subtle changes in several tissues together affects body metabolism or disease progression. This could be applied to improve diets for subjects exposed to health risks associated with obesity.

  14. High-protein goat's milk diet identified through newborn screening: clinical warning of a potentially dangerous dietetic practice.

    Science.gov (United States)

    Maines, Evelina; Gugelmo, Giorgia; Tadiotto, Elisa; Pietrobelli, Angelo; Campostrini, Natascia; Pasini, Andrea; Ion-Popa, Florina; Vincenzi, Monica; Teofoli, Francesca; Camilot, Marta; Bordugo, Andrea

    2017-10-01

    Breast-feeding is an unequalled way of providing optimal food for infants' healthy growth and development and the WHO recommends that infants should be exclusively breast-fed for the first 6 months of life. For mothers who are unable to breast-feed or who decide not to, infant formulas are the safest alternative. Despite recommendations, it is possible that parents make potentially harmful nutritional choices for their children because of cultural beliefs or misinformation on infant nutrition. We describe a possible health risk of not breast-feeding, highlighting a potentially dangerous dietetic practice. Design/Setting/Subjects We report the case of a newborn who was fed with undiluted goat's milk because her mother could not breast-feed and was not aware of infant formulas. The dietary mistake was detected because of a positive expanded newborn screening result, characterized by severe hypertyrosinaemia with high methionine and phenylalanine levels, a pattern suggestive of severe liver impairment. The pattern of plasma amino acids was related to a goat's milk diet, because of its very different composition compared with human milk and infant formula. Our experience demonstrates that, when breast-feeding is not possible or is not exclusive, infants may be at risk of dangerous nutritional practices, including diets with very high protein content, such as a goat's milk diet. Families of not breast-fed infants may need appropriate advice on safe alternatives for infant nutrition to avoid the risks of inappropriate diets.

  15. Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

    Science.gov (United States)

    Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

    2016-12-01

    During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

  16. Global mapping of protein phosphorylation events identifies novel signalling hubs mediating fatty acid starvation responses in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pultz, Dennis; Bennetzen, Martin; Rødkær, Steven Vestergaard

    2011-01-01

    Dietary restriction (DR) extends the life span of multiple species, ranging from single-celled organisms like yeast to mammals. This increase in longevity by dietary restriction is coupled to profound beneficial effects on age-related pathology. Despite the number of studies on DR...... and the physiological changes DR induces, only little is known about the genetics and signalling networks, which regulate the DR response. We have recently shown that inhibition of fatty acid synthesis in Saccharomyces cerevisiae induces autophagy mediated by TORC1 signalling and affects life span. In the present study...... in a temporal manner in response to inhibition of fatty acid synthesis by cerulenin. By in silico analysis of these phosphorylation events, we have identified the major downstream regulated processes and signalling networks mediating the cellular response to fatty acid starvation. The analysis further...

  17. Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I.

    Science.gov (United States)

    Liao, Ming-Xiang; Liu, Dong-Yuan; Zuo, Jin; Fang, Fu-De

    2002-03-01

    To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI). Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. The binding of c-Jun and ANT to GPEI core sequence were confirmed. Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

  18. Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

    Science.gov (United States)

    Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

    2017-06-19

    The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

  19. Pacific Partners Wargame Analysis

    Science.gov (United States)

    2014-09-01

    such as medical qualifications and malpractice considerations, can be explored as part of interoperability. Partner nations are motivated to...prefers a medical research focus. Subsuming medical under HA/DR attempts to minimize those differences. There were dissenting views on the proposed...positive view of USARPAC engagement activities, they were still able to develop ideas for ways to improve engagement activities by modifying or

  20. Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients

    International Nuclear Information System (INIS)

    Tian, Mei; Cui, Ya-Zhou; Song, Guan-Hua; Zong, Mei-Juan; Zhou, Xiao-Yan; Chen, Yu; Han, Jin-Xiang

    2008-01-01

    There is an urgent need to discover more sensitive and specific biomarkers to improve early diagnosis and screen high-risk patients for pancreatic ductal adenocarcinoma (PDAC). Pancreatic juice is an ideal specimen for PDAC biomarkers discovery, because it is an exceptionally rich source of proteins released from pancreatic cancer cells. To identify novel potential biomarkers for PDAC from pancreatic juice, we carried out difference gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS) to compare the pancreatic juice profiling from 9 PDAC patients and 9 cancer-free controls. Of the identified differently expressed proteins, three up-regulated proteins in pancreatic cancer juice, matrix metalloproteinase-9 (MMP-9), oncogene DJ1 (DJ-1) and alpha-1B-glycoprotein precursor (A1BG), were selected for validation by Western blot and immunohistochemistry. Serum MMP-9 levels were also detected by enzyme linked immunosorbent assay (ELISA). Fourteen proteins were up-regulated and ten proteins were down-regulated in cancerous pancreatic juice compared with cancer-free controls. Increased MMP-9, DJ-1 and A1BG expression in cancerous pancreatic juice were confirmed by Western blot. Immunohistochemical study showed MMP-9, DJ-1 and A1BG positively expressed in 82.4%, 72.5% and 86.3% of pancreatic cancer tissues, significantly higher than that in normal pancreas tissues. Up-regulation of DJ-1 was associated with better differentiation (p < 0.05). Serum MMP-9 levels were significantly higher in PDAC (255.14 ng/ml) than those in chronic pancreatitis (210.22 ng/ml, p = 0.009) and healthy control (203.77 ng/ml, p = 0.027). The present proteome analysis revealed MMP-9, DJ-1 and A1BG proteins as elevated in pancreatic juice from PDAC, which suggest their further utility in PDAC diagnosis and screening. This is the first time A1BG was identified as a potential biomarker in pancreatic cancer associated samples. The measurement of serum MMP-9 might be clinically useful for PDAC

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Audit quality and the audit partner effect : Evidence from European listed companies

    OpenAIRE

    Buuren, van, J.P.

    2009-01-01

    The main objective of this study is to provide evidence on the differences in audit quality amongst audit partners. I attribute these dissimilarities to (i) differences in the audit risk perception and the risk appetite of individual audit partners and (ii) to differences in the personal business case of audit partners. As a result, three audit partner archetypes have been identified: liberal, high quality and conservative. This paper will provide evidence that 50% of the audit partners (53% ...

  3. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    Directory of Open Access Journals (Sweden)

    Michael S Rogers

    Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

  4. Interaction Quality during Partner Reading

    OpenAIRE

    Meisinger, Elizabeth B.; Schwanenflugel, Paula J.; Bradley, Barbara A.; Stahl, Steven A.

    2004-01-01

    The influence of social relationships, positive interdependence, and teacher structure on the quality of partner reading interactions was examined. Partner reading, a scripted cooperative learning strategy, is often used in classrooms to promote the development of fluent and automatic reading skills. Forty-three pairs of second grade children were observed during partner reading sessions taking place in 12 classrooms. The degree to which the partners displayed social cooperation (instrumental...

  5. Partner personality in distressed relationships

    NARCIS (Netherlands)

    Barelds, D.P.H.; Barelds-Dijkstra, P.

    2006-01-01

    The present study examines the personality characteristics of partners receiving marital therapy. On the basis of previous research, we expected partners in distressed relationships to be more neurotic and more introverted and to have lower self-esteem than partners in non-distressed relationships.

  6. Nucleolar targeting of proteins by the tandem array of basic amino acid stretches identified in the RNA polymerase I-associated factor PAF49

    International Nuclear Information System (INIS)

    Ushijima, Ryujiro; Matsuyama, Toshifumi; Nagata, Izumi; Yamamoto, Kazuo

    2008-01-01

    There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199-338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence

  7. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    Science.gov (United States)

    Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715

  8. The 1.1 Å resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank.

    Science.gov (United States)

    Keegan, Ronan; Waterman, David G; Hopper, David J; Coates, Leighton; Taylor, Graham; Guo, Jingxu; Coker, Alun R; Erskine, Peter T; Wood, Steve P; Cooper, Jonathan B

    2016-08-01

    During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate

  9. High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

    Science.gov (United States)

    Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

    2014-09-01

    Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Beauty is in the eye of the beholder: proteins can recognize binding sites of homologous proteins in more than one way.

    Directory of Open Access Journals (Sweden)

    Juliette Martin

    2010-06-01

    Full Text Available Understanding the mechanisms of protein-protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein-protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein-protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.

  11. Venues for Meeting Sex Partners and Partner HIV Risk Characteristics: HIV Prevention Trials Network (HPTN064) Women's HIV Seroincidence Study (ISIS).

    Science.gov (United States)

    Roman Isler, M; Golin, C; Wang, J; Hughes, J; Justman, J; Haley, D; Kuo, I; Adimora, A; Chege, W; Hodder, S

    2016-06-01

    Identifying venues where women meet sexual partners, particular partners who increase women's risk of acquiring HIV, could inform prevention efforts. We categorized venues where women enrolled in HPTN 064 reported meeting their last three sex partners as: (1) Formal, (2) Public, (3) Private, and (4) Virtual spaces. We used multinomial logistic regression to assess the association between these venues and women's individual characteristics and reports of their partners' HIV risk characteristics. The 2099 women reported meeting 3991 partners, 51 % at Public, 30 % Private, 17 % Formal and 3 % at Virtual venues. Women meeting partners at Formal venues reported more education and condom use than women meeting partners at other venues. Fewer partners met through Formal venues had "high" risk characteristics for HIV than through other venues and hence may pose less risk of HIV transmission. HIV prevention interventions can help women choose partners with fewer risk characteristics across all venue types.

  12. Care partner: A concept analysis.

    Science.gov (United States)

    Bennett, Paul N; Wang, Wei; Moore, Mel; Nagle, Cate

    The use of the term care partner has increased, particularly in the chronic disease literature; however, the concept has not been well defined. The purpose of this concept analysis was to define and assist nurses to better understand the concept of care partner. The method by Walker and Avant was used for this literature-based concept analysis. Care partnering includes providing assistance to an individual with a health condition to meet their self-care deficits, the commitment to a care partner relationship, and the recognition that people with self-care deficits are care partners contributing to their own care. Emphasizing the care partner dyad in nursing may contribute to improved patient care outcomes both in the acute and chronic settings. It is recommended that nurses view the person with the condition as a contributor and partner in their own care in the context of a larger care partnership. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. New partner - Forestland

    CERN Multimedia

    Staff Association

    2014-01-01

      New partner   Do you need a moment of relaxation and adventure?  Come to Divonne-les-bains and benefit of an immediate discount of 20% on all ropes courses of Forestland http://www.forestland.fr/ upon presentation of your Staff Association membership card. The park is open from 10h00 to 19h00 on Wednesday, Saturday, Sunday during the school period and public holidays (France and Switzerland) or every day during school holidays (France and Switzerland). Different levels of difficulty are available: children, juniors, adults, athletes.

  14. Benefiting through partnering

    International Nuclear Information System (INIS)

    Carr, T.J.

    2000-01-01

    As a consequence of dramatic changes in the world market in nuclear services over the last decade, BNFL has embarked on a comprehensive strategic review of its business. Central to this review has been the need for the company to achieve cost reduction and improved efficiency in all aspects of its business. An area where substantial benefits can be gained is in improved efficiency in the discharge of the capital expenditure programme. This paper focuses on the opportunity of profiting through partnering in capital project delivery. (author)

  15. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    International Nuclear Information System (INIS)

    Xu, H. Howard; Real, Lilian; Bailey, Melissa Wu

    2006-01-01

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats

  16. Tracking micro-optical resonances for identifying and sensing novel procaspase-3 protein marker released from cell cultures in response to toxins

    International Nuclear Information System (INIS)

    Chen, Ying-Jen; Vollmer, Frank; Xiang, Wei; Klucken, Jochen

    2016-01-01

    The response of cells to toxins is commonly investigated by detecting intracellular markers for cell death, such as caspase proteins. This requires the introduction of labels by the permeabilization or complete lysis of cells. Here we introduce a non-invasive tool for monitoring a caspase protein in the extracellular medium. The tool is based on highly sensitive optical micro-devices, referred to as whispering-gallery mode biosensors (WGMBs). WGMBs are functionalized with antibodies for the specific and label-free detection of procaspase-3 released from human embryonic kidney HEK293 and neuroglioma H4 cells after introducing staurosporine and rotenone toxins, respectively. Additional tests show that the extracellular accumulation of procaspase-3 is concomitant with a decrease in cell viability. The hitherto unknown release of procaspase-3 from cells in response to toxins and its accumulation in the medium is further investigated by Western blot, showing that the extracellular detection of procaspase-3 is interrelated with cytotoxicity of alpha-synuclein protein (aSyn) overexpressed in H4 cells. These studies provide evidence for procaspase-3 as a novel extracellular biomarker for cell death, with applications in cytotoxicity tests. Such WGMBs could be applied to further identify as-yet unknown extracellular biomarkers using established antibodies against intracellular antigens. (paper)

  17. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    Directory of Open Access Journals (Sweden)

    J. M. H. Geddes

    2016-01-01

    Full Text Available The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis.

  18. Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

    Directory of Open Access Journals (Sweden)

    Kumagai Yoshinori

    2010-12-01

    Full Text Available Abstract Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium, several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

  19. The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

    Directory of Open Access Journals (Sweden)

    Stéphanie eCottier

    2011-12-01

    Full Text Available Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx. In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA binding domain (encoded in the yeast strain, and the bioactive molecule part binding to its potential protein target fused to a DNA activating domain (encoded on a cDNA expression vector. During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discussed the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

  20. Venues for Meeting Sex Partners and Partner HIV Risk Characteristics: HIV Prevention Trials Network (HPTN064) Women's HIV Seroincidence Study (ISIS)

    OpenAIRE

    Isler, M. Roman; Golin, C.; Wang, J.; Hughes, J.; Justman, J.; Haley, D.; Kuo, I.; Adimora, A.; Chege, W.; Hodder, S.

    2016-01-01

    Identifying venues where women meet sexual partners, particular partners who increase women's risk of acquiring HIV, could inform prevention efforts. We categorized venues where women enrolled in HPTN 064 reported meeting their last three sex partners as: (1) Formal, (2) Public, (3) Private, and (4) Virtual spaces. We used multinomial logistic regression to assess the association between these venues and women's individual characteristics and reports of their partners' HIV risk characteristic...

  1. Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

    Science.gov (United States)

    Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

    2018-04-15

    African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, the