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Sample records for identified sr protein

  1. Negative regulation of active zone assembly by a newly identified SR protein kinase.

    Directory of Open Access Journals (Sweden)

    Ervin L Johnson

    2009-09-01

    Full Text Available Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila or ribbon (vertebrates are believed to facilitate vesicle movement to the active zone (AZ of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D. This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  2. Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

    Science.gov (United States)

    Liu, Boyin; Bossing, Torsten

    2016-01-01

    We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently. PMID:27725692

  3. Advances in the Study of SR Protein Family

    Institute of Scientific and Technical Information of China (English)

    XiaoyunMa; FuchuHe

    2003-01-01

    The name of SR proteins is derived from their typical RS domain that is rich in serine(Ser,S)and arginine(Arg,R).They are conserved in evolution.Up to now,10 members of the SR protein family have been identified in humans.SR proteins contain one or two RNA binding motifs aside from the RS domain,and also possess special biochemical and immunological features.As to the functions of SR proteins,they facilitate the recruitment of the components of splicesome via protein-protein interaction to prompt the assembly ofearly aplicesome;while in alternative splicing,tissue-specifically expressed SR protein along with the relative ratio of SR protein and heterogeneous nuclear ribonucleoprotein(hnRNP)is composed of two main regulative mechanisms for alternative splicing.Almost all of the biochemical functions are regulated by reversible phosphorylation.

  4. Advances in the Study of SR Protein Family

    Institute of Scientific and Technical Information of China (English)

    Xiaoyun Ma; Fuchu He

    2003-01-01

    The name of SR proteins is derived from their typical RS domain that is rich in serine (Ser, S) and arginine (Arg, R). They are conserved in evolution. Up to now, 10 members of the SR protein family have been identified in humans. SR proteins contain one or two RNA binding motifs aside from the RS domain, and also possess special biochemical and immunological features. As to the functions of SR proteins, they facilitate the recruitment of the components of splicesome via protein-protein interaction to prompt the assembly of early splicesome; while in alternative splicing, tissue-specifically expressed SR protein along with the relative ratio of SR protein and heterogeneous nuclear ribonucleoprotein (hnRNP) is composed of two main regulative mechanisms for alternative splicing. Almost all of the biochemical functions are regulated by reversible phosphorylation.

  5. The inverse F-BAR domain protein srGAP2 acts through srGAP3 to modulate neuronal differentiation and neurite outgrowth of mouse neuroblastoma cells.

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    Yue Ma

    Full Text Available The inverse F-BAR (IF-BAR domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1(R542A and srGAP2(R527A exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.

  6. Transitions of protein traffic from cardiac ER to junctional SR

    OpenAIRE

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appe...

  7. Use of 87Sr/86Sr and δ11B to Identify Slag-Affected Sediment in Southern Lake Michigan

    Science.gov (United States)

    Bayless, E. Randall; Bullen, Thomas D.; Fitzpatrick, John A.

    2004-01-01

    Slag is a ubiquitous byproduct of the iron-smelting industry and influences geochemistry and water quality in adjacent geologic units, ground and surface water. Despite extensive slag deposition along the Indiana shoreline of Lake Michigan, definitive evidence that slag has affected lakebed sediments has not been established. Concerns for the protection of water and ecosystem resources in the Great Lakes motivated this study to determine if strontium and boron isotopes could be used to identify and delineate slag-affected bed sediment in Lake Michigan. Sixty-five samples of bed sediment were acquired from the southern lobe of Lake Michigan and analyzed for 87Sr/86Sr and ??11B. Samples immediately offshore from Indiana steel mills and slag-disposal sites contained higher median 87Sr/86Sr values (0.70881) than shoreline sediments collected elsewhere in the basin (0.70847) and uniquely decreased with increasing distance from the shoreline. The highest ??11B values occurred in sediments from the Indiana shoreline (+12.9 to 16.4???) but were also elevated in sediments collected offshore from three Lake Michigan cities (+11.7 to 12.7???). Contoured isotope data indicated that 82-154 km2 of bed sediment along the Indiana shoreline had elevated 87Sr/86Sr and ??11B values relative to shoreline sediments elsewhere in southern Lake Michigan.

  8. Transitions of protein traffic from cardiac ER to junctional SR.

    Science.gov (United States)

    Sleiman, Naama H; McFarland, Timothy P; Jones, Larry R; Cala, Steven E

    2015-04-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca(2+) release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQ-DsRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed ((del)TRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and (del)TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic.

  9. Transitions of protein traffic from cardiac ER to junctional SR

    Science.gov (United States)

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and del TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic. PMID:25640161

  10. Protection against retrovirus pathogenesis by SR protein inhibitors.

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    Anne Keriel

    Full Text Available Indole derivatives compounds (IDC are a new class of splicing inhibitors that have a selective action on exonic splicing enhancers (ESE-dependent activity of individual serine-arginine-rich (SR proteins. Some of these molecules have been shown to compromise assembly of HIV infectious particles in cell cultures by interfering with the activity of the SR protein SF2/ASF and by subsequently suppressing production of splicing-dependent retroviral accessory proteins. For all replication-competent retroviruses, a limiting requirement for infection and pathogenesis is the expression of the envelope glycoprotein which strictly depends on the host splicing machinery. Here, we have evaluated the efficiency of IDC on an animal model of retroviral pathogenesis using a fully replication-competent retrovirus. In this model, all newborn mice infected with a fully replicative murine leukemia virus (MLV develop erythroleukemia within 6 to 8 weeks of age. We tested several IDC for their ability to interfere ex vivo with MLV splicing and virus spreading as well as for their protective effect in vivo. We show here that two of these IDC, IDC13 and IDC78, selectively altered splicing-dependent production of the retroviral envelope gene, thus inhibiting early viral replication in vivo, sufficiently to protect mice from MLV-induced pathogenesis. The apparent specificity and clinical safety observed here for both IDC13 and IDC78 strongly support further assessment of inhibitors of SR protein splicing factors as a new class of antiretroviral therapeutic agents.

  11. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

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    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  12. A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2.

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    Liu, Shide; Kang, Kang; Zhang, Jianhua; Ouyang, Qiuling; Zhou, Zhuolong; Tian, Shengli; Xing, Miao

    2009-08-01

    A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

  13. A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2

    Institute of Scientific and Technical Information of China (English)

    Shide Liu; Kang Kang; Jianhua Zhang; Qiuling Ouyang; Zhuolong Zhou; Shengli Tian; Miao Xing

    2009-01-01

    A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-iike protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homolo-gous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-term-inal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphoryi-ation assay to investigate the phospborylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Auto-radiographic examination showed that both recombi-nant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could pbosphorylate ASF/SF2 and a truncated form of ASF/SF2 that con-tains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and func-tion of PSRPK demonstrates that it is a new member of the SRPK family.

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

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    Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

    2012-10-01

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

  15. Identifying Protein-Calorie Malnutrition Workshop.

    Science.gov (United States)

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  16. Ontology integration to identify protein complex in protein interaction networks

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    Yang Zhihao

    2011-10-01

    Full Text Available Abstract Background Protein complexes 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 protein complexes detection algorithms. Methods We have developed novel semantic similarity method, 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. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches.

  17. Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

    Institute of Scientific and Technical Information of China (English)

    杨仲安; 曹丹; 桂建芳

    2000-01-01

    The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

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

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    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

  19. Targeting SR proteins improves SMN expression in spinal muscular atrophy cells.

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    Claribel D Wee

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

  20. Cellular differentiation state modulates the mRNA export activity of SR proteins.

    Science.gov (United States)

    Botti, Valentina; McNicoll, François; Steiner, Michaela C; Richter, Florian M; Solovyeva, Anfisa; Wegener, Marius; Schwich, Oliver D; Poser, Ina; Zarnack, Kathi; Wittig, Ilka; Neugebauer, Karla M; Müller-McNicoll, Michaela

    2017-07-03

    SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells. © 2017 Botti et al.

  1. InterProScan: protein domains identifier.

    Science.gov (United States)

    Quevillon, E; Silventoinen, V; Pillai, S; Harte, N; Mulder, N; Apweiler, R; Lopez, R

    2005-07-01

    InterProScan [E. M. Zdobnov and R. Apweiler (2001) Bioinformatics, 17, 847-848] is a tool that combines different protein signature recognition methods from the InterPro [N. J. Mulder, R. Apweiler, T. K. Attwood, A. Bairoch, A. Bateman, D. Binns, P. Bradley, P. Bork, P. Bucher, L. Cerutti et al. (2005) Nucleic Acids Res., 33, D201-D205] consortium member databases into one resource. At the time of writing there are 10 distinct publicly available databases in the application. Protein as well as DNA sequences can be analysed. A web-based version is accessible for academic and commercial organizations from the EBI (http://www.ebi.ac.uk/InterProScan/). In addition, a standalone Perl version and a SOAP Web Service [J. Snell, D. Tidwell and P. Kulchenko (2001) Programming Web Services with SOAP, 1st edn. O'Reilly Publishers, Sebastopol, CA, http://www.w3.org/TR/soap/] are also available to the users. Various output formats are supported and include text tables, XML documents, as well as various graphs to help interpret the results.

  2. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes.

    Science.gov (United States)

    Änkö, Minna-Liisa; Müller-McNicoll, Michaela; Brandl, Holger; Curk, Tomaz; Gorup, Crtomir; Henry, Ian; Ule, Jernej; Neugebauer, Karla M

    2012-01-01

    The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells. SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay. SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members.

  3. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    Science.gov (United States)

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

  4. Is otolith microchemistry (Sr: Ca and Ba:Ca ratios useful to identify Mugil curema populations in the southeastern Caribbean Sea?

    Directory of Open Access Journals (Sweden)

    E. Avigliano

    Full Text Available Abstract The aim of the present study was to evaluate the potential use of otolith microchemistry (Sr:Ca and Ba:Ca ratios to identify silver mullet, Mugil curema, populations in Southeastern Caribbean Sea. Fish samples were collected in 7 areas of Nueva Esparta State (Venezuela. The otolith Sr:Ca and Ba:Ca ratios and water Sr:Ca were determined (by ICP-OES and EDTA volumetric method. Otoliths Sr:Ca and Ba:Ca ratios and Sr:Ca partition coefficient of mullets in Cubagua island (south of the State were significantly different from ratios in La Guardia (north of the State. A discriminant analysis of otolith Sr:Ca and Ba:Ca ratios separated Cubagua Island from La Guardia values. These results suggest the existence of different mullet groups in the Southeastern Caribbean Sea. For this, the simultaneous use of Sr:Ca and Ba:Ca ratios could be a potential tool to identify populations in the study area.

  5. Trace elemental and 87Sr/86Sr isotopic compositions as a method for identifying sediment sources in a major coastal estuary

    Science.gov (United States)

    Malamud-Roam, F. P.; Ingram, B. L.; Malamud-Roam, K. P.; Collins, J.

    2005-12-01

    Maintenance and health of tidal wetlands depends most fundamentally on adequate sediment supply. In the San Francisco Bay estuary, tidal marshes that once surrounded the estuary providing natural protection from storms, habitat for a variety of plant and animal species and a host of other important ecological functions, are threatened by rising sea level and insufficient sediments. We present results from an ongoing study evaluating the relative sources of sediments that feed the San Francisco Bay tidal marshlands using their geochemical signatures. We have analyzed the concentrations of 21 trace and major elements and 87Sr/86Sr isotopic ratios in the suspended sediments of major tributaries to the Sacramento - San Joaquin Delta and of smaller tributaries surrounding the northern reach of the Bay estuary. We have also analyzed the geochemical compositions of sediments from marsh surface samples and from a series of short (50-100 cm long) sediment cores collected from Novato Creek Marsh, on the western edge of San Pablo bay. Previous results show that the sediments from the three primary end members can be distinguished, and the results presented here will elaborate, using a combination of multivariate analyses and a mixing model to evaluate the likely sources of sediments occupying the marsh surface, both modern and pre- modern (i.e., before large-scale changes to California hydrologic systems). Initial elemental and 87Sr/86Sr isotopic results indicate that a significant share of the suspended sediments in the estuary and on the marsh surfaces may not come from either of the two major river systems, the Sacramento and San Joaquin. The mean 87Sr/86Sr isotopic ratio measured in samples from Sacramento river tributaries is 0. 706380 and the measured mean of samples from the San Joaquin river tributaries is 0.707191. Of the samples collected from local creeks around San Pablo bay that have been analyzed to date, there is a range of Sr isotope values from 0.709243 to 0

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

    Energy Technology Data Exchange (ETDEWEB)

    A.S.N. Reddy

    2008-11-25

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

  7. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes

    OpenAIRE

    Neugebauer, Karla M.; Änkö, Minna-Liisa; Müller-McNicoll, Michaela; Brandl, Holger; Henry, Ian; Gorup, Črtomir; Curk, Tomaž; Ule, Jernej

    2015-01-01

    Background The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells. Results SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intro...

  8. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    Science.gov (United States)

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina; Norbeck, Angela D.; Qian, Wei-Jun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-01

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics applying LC-MS/MS and ELISA to analyze a set of 92urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after kidney transplantation. PMID:20543976

  9. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    Energy Technology Data Exchange (ETDEWEB)

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina A.; Norbeck, Angela D.; Qian, Weijun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-04

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics using LC-MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after

  10. Male-specific phosphorylated SR proteins in adult flies of the Mediterranean fruitfly Ceratitis capitata.

    Science.gov (United States)

    Saccone, Giuseppe; Louis, Christos; Zhang, Hongyou; Petrella, Valeria; Di Natale, Manuela; Perri, Maria; Salvemini, Marco

    2014-01-01

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

  11. The use of O, H and Sr isotopes and carbamazepine to identify the origin of water bodies supplying a shallow alluvial aquifer

    Science.gov (United States)

    Sassine, Lara; Le Gal La Salle, Corinne; Lancelot, Joël; Verdoux, Patrick

    2014-05-01

    Alluvial aquifers are of great socio-economic importance in France since they supply 82% of drinking water production, though they reveal to be very vulnerable to pesticides and emerging organic contaminants. The aim of this work is to identify the origin of water bodies which contribute to the recharge of an alluvial aquifer for a better understanding of its hydrochemistry and transfer of contaminants therein. The study is based on an isotopic and geochemical tracers approach, including major elements, trace elements (Br, Sr),and isotopes (δ18O, δ2H, 87Sr/86Sr), as well as organic molecules. Indeed, organic molecules such as pharmaceutical compounds, more precisely carbamazepine and caffeine, have shown their use as indicators of surface water in groundwater. The study area is a partially-confined shallow alluvial aquifer, the so-called Vistrenque aquifer, located at 15 km from the Mediterranean Sea, in the Quaternary alluviums deposited by an ancient arm of the Rhône River, in Southern France. This aquifer constitutes a shallow alluvial layer in a NE-SW graben structure. It is situated between a karst aquifer in lower Cretaceous limestones, on the NW border, and the Costières Plateau, on the SE border, having a similar geology as the Vistrenque. The alluvial plain is crossed by a surface water network with the Vistre as the main stream, and a canal used for irrigation essentially, the BRL canal, which is fed by the Rhône River. δ18O and δ2H allowed to differentiate the BRL canal water, depleted in heavy isotopes (δ2H = -71.5o vs V-SMOW), and the more enriched local rainwater (δ2H = -35.5o vs V-SMOW). In the Vistre surface water a binary mixing were evidenced with the BRL canal water and the rainwater, as end members. Then, in the Vistrenque groundwater both the BRL and the Vistre contributions could be identified, as they still show contrasting signature with local recharge. This allows to highlight the surface water contribution to a heavily exploited

  12. Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway.

    Science.gov (United States)

    Ote, Isabelle; Lebrun, Marielle; Vandevenne, Patricia; Bontems, Sébastien; Medina-Palazon, Cahora; Manet, Evelyne; Piette, Jacques; Sadzot-Delvaux, Catherine

    2009-11-18

    Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

  13. Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway.

    Directory of Open Access Journals (Sweden)

    Isabelle Ote

    Full Text Available Available data suggest that the Varicella-Zoster virus (VZV IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

  14. A least square method based model for identifying protein complexes in protein-protein interaction network.

    Science.gov (United States)

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

    2014-01-01

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

  15. Identifying folding nucleus based on residue contact networks of proteins.

    Science.gov (United States)

    Li, Jie; Wang, Jun; Wang, Wei

    2008-06-01

    In the native structure of a protein, all the residues are tightly parked together in a specific order following its folding and every residue contacts with some spatially neighbor residues. A residue contact network can be constructed by defining the residues as nodes and the native contacts as edges. During the folding of small single-domain proteins, there is a set of contacts (or bonds), defined as the folding nucleus (FN), which is formed around the transition state, i.e., a rate-limiting barrier located at about the middle between the unfolded states and the native state on the free energy landscape. Such a FN plays an essential role in the folding dynamics and the residues, which form the related contacts called as folding nucleus residues (FNRs). In this work, the FNRs in proteins are identified by using quantities which characterize the topology of residue contact networks of proteins. By comparing the specificities of residues with the network quantities K(R), L(R), and D(R), up to 90% FNRs of six typical proteins found experimentally are identified. It is found that the FNRs behave the full-closeness centrals rather than degree or closeness centers in the residue contact network, implying that they are important to the folding cooperativity of proteins. Our study shows that the FNRs can be identified solely from the native structures of proteins based on the analysis of residue contact network without any knowledge of the transition state ensemble. (c) 2008 Wiley-Liss, Inc.

  16. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Science.gov (United States)

    2010-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1 are...

  17. Proteins identified from care solution extractions of silicone hydrogels.

    Science.gov (United States)

    Emch, Andrew J; Nichols, Jason J

    2009-02-01

    The purpose of this study was to investigate the quantity and identify the proteins extracted from two different types of silicone hydrogel contact lenses by several multipurpose care solutions after 1 day of wear. Ten subjects were recruited to wear galyfilcon A lenses (Acuvue Advance, Vistakon) followed by lotrafilcon B lenses (O2 Optix, CIBA Vision) each for four consecutive days. Each day, subjects inserted a new pair of lenses for 8 h of wear after which both lenses were removed using forceps (lenses were not rubbed or rinsed after removal). Lenses were pooled in one of four commercially available care solutions for a 24-h soak followed by precipitation, resuspension in water, and quantification by Bradford assay and identification by mass spectrometry. Protein recovery from care solutions was as follows (quantities are in microg/lens): AQuify (galyfilcon A: 0.56, lotrafilcon B: 1.24), Complete MoisturePlus (galyfilcon A: 1.44, lotrafilcon B: 1.47), Opti-Free Express (galyfilcon A: 2.31, lotrafilcon B: 5.67), and ReNu MoistureLoc (galyfilcon A: 1.17, lotrafilcon B: 4.38). For each care solution, greater quantities of protein were removed from lotrafilcon B (3.19 +/- 2.19 microg/lens) than from galyfilcon A (1.37 +/- 0.72 microg/lens). Lactoferrin, lysozyme, and lipocalin were the most commonly identified, whereas various keratin compounds and other unique proteins were also detected. Opti-Free Express was consistently associated with the more efficient removal of proteins from these silicone hydrogels. More total protein was removed from lotrafilcon B than from galyfilcon A (approximately 2 x more protein) for all four care solutions, and 12 total unique protein species were recovered from galyfilcon A, whereas only 10 were recovered from lotrafilcon B. The higher quantities of protein extracted from lotrafilcon B may be due to stronger protein binding with this material and/or to differences in solution efficacy.

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

    Science.gov (United States)

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

    2013-01-01

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

  19. Dissecting the regulon of the two-component system CvsSR: Identifying new virulence genes in Pseudomonas syringae pv. tomato DC3000

    Science.gov (United States)

    Recognition of environmental changes and regulation of genes that allow for adaption to those changes is essential for survival of bacteria. Two-component systems (TCSs) allow bacteria to sense and adapt to their environment. We previously identified the TCS CvsSR in the bacterial plant pathogen Pse...

  20. 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.

  1. 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/.

  2. Identifying unexpected therapeutic targets via chemical-protein interactome.

    Directory of Open Access Journals (Sweden)

    Lun Yang

    Full Text Available Drug medications inevitably affect not only their intended protein targets but also other proteins as well. In this study we examined the hypothesis that drugs that share the same therapeutic effect also share a common therapeutic mechanism by targeting not only known drug targets, but also by interacting unexpectedly on the same cryptic targets. By constructing and mining an Alzheimer's disease (AD drug-oriented chemical-protein interactome (CPI using a matrix of 10 drug molecules known to treat AD towards 401 human protein pockets, we found that such cryptic targets exist. We recovered from CPI the only validated therapeutic target of AD, acetylcholinesterase (ACHE, and highlighted several other putative targets. For example, we discovered that estrogen receptor (ER and histone deacetylase (HDAC, which have recently been identified as two new therapeutic targets of AD, might already have been targeted by the marketed AD drugs. We further established that the CPI profile of a drug can reflect its interacting character towards multi-protein sets, and that drugs with the same therapeutic attribute will share a similar interacting profile. These findings indicate that the CPI could represent the landscape of chemical-protein interactions and uncover "behind-the-scenes" aspects of the therapeutic mechanisms of existing drugs, providing testable hypotheses of the key nodes for network pharmacology or brand new drug targets for one-target pharmacology paradigm.

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

    Directory of Open Access Journals (Sweden)

    Giulia Bonente

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

  4. Isolated pseudo-RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition.

    Science.gov (United States)

    Cléry, Antoine; Sinha, Rahul; Anczuków, Olga; Corrionero, Anna; Moursy, Ahmed; Daubner, Gerrit M; Valcárcel, Juan; Krainer, Adrian R; Allain, Frédéric H-T

    2013-07-23

    Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.

  5. CYP1A-immunopositive proteins in bivalves identified as cytoskeletal and major vault proteins

    DEFF Research Database (Denmark)

    Grøsvik, Bjørn Einar; Jonsson, Henrik; Rodríguez-Ortega, Manuel J;

    2006-01-01

    To identify possible CYP1A-immunopositive proteins in bivalves, we used anti-fish CYP1A antibodies combined with one- and two-dimensional gel electrophoresis and mass spectrometry, and found that two of the main CYP1A-immunopositive proteins in digestive gland of Mytilus edulis, were cytoskeletal...

  6. A new scoring function for protein-protein docking that identifies native structures with unprecedented accuracy

    DEFF Research Database (Denmark)

    Moreira, Irina S.; da Silva Martins, João Miguel; Coimbra, João T.S.

    2015-01-01

    Protein-protein (P-P) 3D structures are fundamental to structural biology and drug discovery. However, most of them have never been determined. Many docking algorithms were developed for that purpose, but they have a very limited accuracy in generating native-like structures and identifying...... the most correct one, in particular when a single answer is asked for. With such a low success rate it is difficult to point out one docked structure as being native-like. Here we present a new, high accuracy, scoring method to identify the 3D structure of P-P complexes among a set of trial poses...... the trial structures and identifies the native-like structures with unprecedented accuracy (∼94%), providing the correct P-P 3D structures that biochemists and molecular biologists need to pursue their studies. With such a success rate, the bottleneck of protein-protein docking moves from the scoring...

  7. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry

    Directory of Open Access Journals (Sweden)

    Alexandra Traister

    2016-06-01

    Full Text Available Using hearts from mice overexpressing integrin linked kinase (ILK behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD001053. The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  8. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions*

    Science.gov (United States)

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary E.; Geller, Jil T.; Fisher, Susan J.; Hall, Steven C.; Hazen, Terry C.; Brenner, Steven E.; Butland, Gareth; Jin, Jian; Witkowska, H. Ewa; Chandonia, John-Marc; Biggin, Mark D.

    2016-01-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR. PMID:27099342

  9. Identifying true protein complex constituents in interaction proteomics: the example of the DMXL2 protein complex.

    Science.gov (United States)

    Li, Ka Wan; Chen, Ning; Klemmer, Patricia; Koopmans, Frank; Karupothula, Ramesh; Smit, August B

    2012-08-01

    A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Identifying drug-target proteins based on network features

    Institute of Scientific and Technical Information of China (English)

    ZHU MingZhu; GAO Lei; LI Xia; LIU ZhiCheng

    2009-01-01

    Proteins rarely function in isolation Inside and outside cells, but operate as part of a highly Intercon-nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action In terms of informatice. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac-tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins In the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem-onstrated to be drug-target proteins in the literature.

  11. Identifying drug-target proteins based on network features

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Proteins rarely function in isolation inside and outside cells, but operate as part of a highly intercon- nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action in terms of informatics. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac- tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins in the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem- onstrated to be drug-target proteins in the literature.

  12. Restoration of high-density lipoprotein levels by cholesteryl ester transfer protein expression in scavenger receptor class B type I (SR-BI) knockout mice does not normalize pathologies associated with SR-BI deficiency

    NARCIS (Netherlands)

    Hildebrand, Reeni B; Lammers, Bart; Meurs, Illiana; Korporaal, Suzanne J A; De Haan, Willeke; Zhao, Ying; Kruijt, J Kar; Praticò, Domenico; Schimmel, Alinda W M; Holleboom, Adriaan G; Hoekstra, Menno; Kuivenhoven, Jan Albert; Van Berkel, Theo J C; Rensen, Patrick C N; Van Eck, Miranda

    OBJECTIVE: Disruption of scavenger receptor class B type I (SR-BI) in mice impairs high-density lipoprotein (HDL)-cholesterol (HDL-C) delivery to the liver and induces susceptibility to atherosclerosis. In this study, it was investigated whether introduction of cholesteryl ester transfer protein

  13. 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.

  14. Informatics View on the Challenges of Identifying Missing Proteins from Shotgun Proteomics.

    Science.gov (United States)

    Choong, Wai-Kok; Chang, Hui-Yin; Chen, Ching-Tai; Tsai, Chia-Feng; Hsu, Wen-Lian; Chen, Yu-Ju; Sung, Ting-Yi

    2015-12-04

    Protein experiment evidence at protein level from mass spectrometry and antibody experiments are essential to characterize the human proteome. neXtProt (2014-09 release) reported 20 055 human proteins, including 16 491 proteins identified at protein level and 3564 proteins unidentified. Excluding 616 proteins at uncertain level, 2948 proteins were regarded as missing proteins. Missing proteins were unidentified partially due to MS limitations and intrinsic properties of proteins, for example, only appearing in specific diseases or tissues. Despite such reasons, it is desirable to explore issues affecting validation of missing proteins from an "ideal" shotgun analysis of human proteome. We thus performed in silico digestions on the human proteins to generate all in silico fully digested peptides. With these presumed peptides, we investigated the identification of proteins without any unique peptide, the effect of sequence variants on protein identification, difficulties in identifying olfactory receptors, and highly similar proteins. Among all proteins with evidence at transcript level, G protein-coupled receptors and olfactory receptors, based on InterPro classification, were the largest families of proteins and exhibited more frequent variants. To identify missing proteins, the above analyses suggested including sequence variants in protein FASTA for database searching. Furthermore, evidence of unique peptides identified from MS experiments would be crucial for experimentally validating missing proteins.

  15. RNA-protein complexes identified by crosslinking of polysomes.

    Science.gov (United States)

    Sköld, S E

    1981-01-01

    The bifunctional cleavable reagent diepoxybutane was used to investigate the crosslinking of proteins to the 16S and 23S RNA in Escherichia coli ribosomes. The crosslinking patterns from polysomes, accumulated in the absence and presence of oxytetracycline, as well as reassociated 70S ribosomes were compared. The 30S proteins: S3, S4, S5, S7, S8, S9, S12, S13, S14 and S18 were recovered crosslinked to the 16S RNA and the 50S: proteins L1, L2, L4, L13, L14-L21, L15, L16, L17, L18-L23, L19-22-24, L27 and L28 were recovered crosslinked to the 23S RNA, in all three associated states. Proteins crosslinked to the RNA of the heterologous subunit and therefore considered to be at or near the ribosomal subunit interface were, for all three states, proteins S1, S4, S6, S9, S12, S13, S14 and S18 from the small subunit and proteins L16, L17, L20 and L27 from the large subunit. Finally, the recovery of intrasubunit crosslinks was measured for the isolated subunits. Additional crosslinked complexes were observed between 16S RNA and S1, S2 as well as S6 from the 30S subunit; and between 23S RNA and L10, L11, L7/12 from the 50S subunit.

  16. Unique role of SRSF2 in transcription activation and diverse functions of the SR and hnRNP proteins in gene expression regulation.

    Science.gov (United States)

    Mo, Sudong; Ji, Xiong; Fu, Xiang-Dong

    2013-01-01

    Transcription pause release from gene promoters has been recognized to be a critical point for transcriptional regulation in higher eukaryotes. Recent studies suggest that regulatory RNAs are extensively involved in transcriptional control, which may enlist various RNA binding proteins. We recently showed a key role of SRSF2, a member of the SR family of splicing regulators, in binding to promoter-associated small RNA to mediate transcription pause release, a regulatory strategy akin to the function of the HIV Tat protein via binding to the TAR element in nascent RNA to activate transcription. In this report, we further dissect the structural requirement for SRSF2 to function as a transcription activator and extend the analysis to multiple SR and hnRNP proteins by using the MS2 tethering strategy. Our results reveal that SRSF2 is a unique SR protein that activates transcription in a position-dependent manner while three other SR proteins enhance translation in a position-independent fashion. In contrast, multiple hnRNP proteins appear to negatively influence mRNA levels, especially when tethered in the gene body. These findings suggest broad participation of RNA binding proteins in diverse aspects of regulated gene expression at both the transcriptional and posttranscriptional levels in mammalian cells.

  17. Identifying Protein Stabilizing Ligands Using GroEL

    Science.gov (United States)

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  18. Serum Profiling Using Protein Microarrays to Identify Disease Related Antigens

    OpenAIRE

    2014-01-01

    Disease related antigens are of great importance in the clinic. They are used as markers to screen patients for various forms of cancer, to monitor response to therapy, or to serve as therapeutic targets (Chapman et al., Ann Oncol 18(5):868–873, 2007; Soussi et al., Cancer Res 60:1777–1788, 2000; Anderson and LaBaer, J Proteome Res 4:1123–1133, 2005; Levenson, Biochim Biophy Acta 1770:847–856, 2007). In cancer endogenous levels of protein expression may be disrupted or proteins may be express...

  19. 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.

  20. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    Science.gov (United States)

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  1. Identifying Protein Stabilizing Ligands Using GroEL

    OpenAIRE

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges,Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the pote...

  2. Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei

    Science.gov (United States)

    Levy, Gabriela V.; Moretti, Georgina; Tekiel, Valeria S.; Sánchez, Daniel O.

    2015-01-01

    Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family. PMID:26284933

  3. 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.

  4. Bioinformatic approaches to identifying and classifying Rab proteins.

    Science.gov (United States)

    Diekmann, Yoan; Pereira-Leal, José B

    2015-01-01

    The bioinformatic annotation of Rab GTPases is important, for example, to understand the evolution of the endomembrane system. However, Rabs are particularly challenging for standard annotation pipelines because they are similar to other small GTPases and form a large family with many paralogous subfamilies. Here, we describe a bioinformatic annotation pipeline specifically tailored to Rab GTPases. It proceeds in two steps: first, Rabs are distinguished from other proteins based on GTPase-specific motifs, overall sequence similarity to other Rabs, and the occurrence of Rab-specific motifs. Second, Rabs are classified taking either a more accurate but slower phylogenetic approach or a slightly less accurate but much faster bioinformatic approach. All necessary steps can either be performed locally or using the referenced online tools. An implementation of a slightly more involved version of the pipeline presented here is available at RabDB.org.

  5. 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.

  6. 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.

  7. Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting, E-mail: qixiaoting@cnu.edu.cn

    2014-12-12

    Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

  8. A Topology Potential-Based Method for Identifying Essential Proteins from PPI Networks.

    Science.gov (United States)

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

    2015-01-01

    Essential proteins are indispensable for cellular life. It is of great significance to identify essential proteins that can help us understand the minimal requirements for cellular life and is also very important for drug design. However, identification of essential proteins based on experimental approaches are typically time-consuming and expensive. With the development of high-throughput technology in the post-genomic era, more and more protein-protein interaction data can be obtained, which make it possible to study essential proteins from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. Most of these topology based essential protein discovery methods were to use network centralities. In this paper, we investigate the essential proteins' topological characters from a completely new perspective. To our knowledge it is the first time that topology potential is used to identify essential proteins from a protein-protein interaction (PPI) network. The basic idea is that each protein in the network can be viewed as a material particle which creates a potential field around itself and the interaction of all proteins forms a topological field over the network. By defining and computing the value of each protein's topology potential, we can obtain a more precise ranking which reflects the importance of proteins from the PPI network. The experimental results show that topology potential-based methods TP and TP-NC outperform traditional topology measures: degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), subgraph centrality (SC), eigenvector centrality (EC), information centrality (IC), and network centrality (NC) for predicting essential proteins. In addition, these centrality measures are improved on their performance for identifying essential proteins in biological network when controlled by topology potential.

  9. A new computational strategy for identifying essential proteins based on network topological properties and biological information.

    Science.gov (United States)

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2017-01-01

    Essential proteins are the proteins that are indispensable to the survival and development of an organism. Deleting a single essential protein will cause lethality or infertility. Identifying and analysing essential proteins are key to understanding the molecular mechanisms of living cells. There are two types of methods for predicting essential proteins: experimental methods, which require considerable time and resources, and computational methods, which overcome the shortcomings of experimental methods. However, the prediction accuracy of computational methods for essential proteins requires further improvement. In this paper, we propose a new computational strategy named CoTB for identifying essential proteins based on a combination of topological properties, subcellular localization information and orthologous protein information. First, we introduce several topological properties of the protein-protein interaction (PPI) network. Second, we propose new methods for measuring orthologous information and subcellular localization and a new computational strategy that uses a random forest prediction model to obtain a probability score for the proteins being essential. Finally, we conduct experiments on four different Saccharomyces cerevisiae datasets. The experimental results demonstrate that our strategy for identifying essential proteins outperforms traditional computational methods and the most recently developed method, SON. In particular, our strategy improves the prediction accuracy to 89, 78, 79, and 85 percent on the YDIP, YMIPS, YMBD and YHQ datasets at the top 100 level, respectively.

  10. Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+

    Directory of Open Access Journals (Sweden)

    Kumar Shivesh

    2012-08-01

    Full Text Available Abstract Background EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins. Results In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1. The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven. Conclusion Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the

  11. Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

    Science.gov (United States)

    Mullen, Lisa; Seavill, Miles; Hammouz, Raneem; Bottazzi, Barbara; Chan, Philippe; Vaudry, David; Ghezzi, Pietro

    2015-09-29

    Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques.

  12. 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.

  13. Proteomic analysis of cell lines to identify the irinotecan resistance proteins

    Indian Academy of Sciences (India)

    Xing-Chen Peng; Feng-Ming Gong; Meng Wei; X I Chen; Y E Chen; K E Cheng; Feng Gao; Feng Xu; FENG Bi; Ji-Yan Liu

    2010-12-01

    Chemotherapeutic drug resistance is a frequent cause of treatment failure in colon cancer patients. Several mechanisms have been implicated in drug resistance. However, they are not sufficient to exhaustively account for this resistance emergence. In this study, two-dimensional gel electrophoresis (2-DE) and the PDQuest software analysis were applied to compare the differential expression of irinotecan-resistance-associated protein in human colon adenocarcinoma LoVo cells and irinotecan-resistant LoVo cells (LoVo/irinotecan). The differential protein dots were excised and analysed by ESI-Q-TOF mass spectrometry (MS). Fifteen proteins were identified, including eight proteins with decreased expression and seven proteins with increased expression. The identified known proteins included those that function in diverse biological processes such as cellular transcription, cell apoptosis, electron transport/redox regulation, cell proliferation/differentiation and retinol metabolism pathways. Identification of such proteins could allow improved understanding of the mechanisms leading to the acquisition of chemoresistance.

  14. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    Science.gov (United States)

    Tirupula, Kalyan C; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  15. Complex evolutionary relationships among four classes of modular RNA-binding splicing regulators in eukaryotes: the hnRNP, SR, ELAV-like and CELF proteins.

    Science.gov (United States)

    Tang, Yue Hang; Han, Siew Ping; Kassahn, Karin S; Skarshewski, Adam; Rothnagel, Joseph A; Smith, Ross

    2012-12-01

    Alternative RNA splicing in multicellular organisms is regulated by a large group of proteins of mainly unknown origin. To predict the functions of these proteins, classification of their domains at the sequence and structural level is necessary. We have focused on four groups of splicing regulators, the heterogeneous nuclear ribonucleoprotein (hnRNP), serine-arginine (SR), embryonic lethal, abnormal vision (ELAV)-like, and CUG-BP and ETR-like factor (CELF) proteins, that show increasing diversity among metazoa. Sequence and phylogenetic analyses were used to obtain a broader understanding of their evolutionary relationships. Surprisingly, when we characterised sequence similarities across full-length sequences and conserved domains of ten metazoan species, we found some hnRNPs were more closely related to SR, ELAV-like and CELF proteins than to other hnRNPs. Phylogenetic analyses and the distribution of the RRM domains suggest that these proteins diversified before the last common ancestor of the metazoans studied here through domain acquisition and duplication to create genes of mixed evolutionary origin. We propose that these proteins were derived independently rather than through the expansion of a single protein family. Our results highlight inconsistencies in the current classification system for these regulators, which does not adequately reflect their evolutionary relationships, and suggests that a domain-based classification scheme may have more utility.

  16. Identifying functional modules in protein-protein interaction networks: An integrated exact approach

    NARCIS (Netherlands)

    Dittrich, M.; Klau, G.W.; Rosenwald, A.; Dandekar, T.; et al, not CWI

    2008-01-01

    Motivation: With the exponential growth of expression and protein-protein interaction (PPI) data, the frontier of research in system biology shifts more and more to the integrated analysis of these large datasets. Of particular interest is the identification of functional modules in PPI networks, sh

  17. 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.

  18. 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.

  19. Influence of the oxidation state of SrTiO3 plasmas for stoichiometric growth of pulsed laser deposition films identified by laser induced fluorescence

    Directory of Open Access Journals (Sweden)

    Kasper Orsel

    2015-10-01

    Full Text Available By applying two-dimensional laser induced fluorescence (LIF on multiple plasma constituents, we are able to directly link the oxidation of plasma species in a SrTiO3 plasma for pulsed laser deposition to the stoichiometry and quality of the thin films grown. With spatiotemporal LIF mapping of the plasma species in different background gas compositions, we find that Ti and Sr have to be fully oxidized for a stoichiometric growth of crystalline thin films, which gives new input for modeling surface growth, as well as provides additional control over the exact degree of stoichiometry of thin films.

  20. Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

    Science.gov (United States)

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

    2009-01-16

    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-gamma (PPARgamma), 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 PPARgamma as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  1. Transgelin: an androgen-dependent protein identified in the seminal vesicles of three Saharan rodents.

    Science.gov (United States)

    Kaci-Ouchfoun, Naïma; Izemrane, Djamila; Boudrissa, Abdelkrim; Gernigon, Thérèse; Khammar, Farida; Exbrayat, Jean Marie

    2013-10-15

    During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.

  2. Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation.

    Science.gov (United States)

    Kolbach-Mandel, A M; Mandel, N S; Cohen, S R; Kleinman, J G; Ahmed, F; Mandel, I C; Wesson, J A

    2017-04-01

    Drug-related kidney stones are a diagnostic problem, since they contain a large matrix (protein) fraction and are frequently incorrectly identified as matrix stones. A urine proteomics study patient produced a guaifenesin stone during her participation, allowing us to both correctly diagnose her disease and identify proteins critical to this drug stone-forming process. The patient provided three random midday urine samples for proteomics studies; one of which contained stone-like sediment with two distinct fractions. These solids were characterized with optical microscopy and Fourier transform infrared spectroscopy. Immunoblotting and quantitative mass spectrometry were used to quantitatively identify the proteins in urine and stone matrix. Infrared spectroscopy showed that the sediment was 60 % protein and 40 % guaifenesin and its metabolite guaiacol. Of the 156 distinct proteins identified in the proteomic studies, 49 were identified in the two stone-components with approximately 50 % of those proteins also found in this patient's urine. Many proteins observed in this drug-related stone have also been reported in proteomic matrix studies of uric acid and calcium containing stones. More importantly, nine proteins were highly enriched and highly abundant in the stone matrix and 8 were reciprocally depleted in urine, suggesting a critical role for these proteins in guaifenesin stone formation. Accurate stone analysis is critical to proper diagnosis and treatment of kidney stones. Many matrix proteins were common to all stone types, but likely not related to disease mechanism. This protocol defined a small set of proteins that were likely critical to guaifenesin stone formation based on their high enrichment and high abundance in stone matrix, and it should be applied to all stone types.

  3. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    Science.gov (United States)

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  4. Using an isolated rat kidney model to identify kidney origin proteins in urine.

    Directory of Open Access Journals (Sweden)

    Lulu Jia

    Full Text Available The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.

  5. Proteomic Study Identifies Proteins Involved in Brassinosteroid Regulation of Rice Growth

    Institute of Scientific and Technical Information of China (English)

    Fengru Wang; Ming-Yi Bai; Zhiping Deng; Juan A. Oses-Prieto; Alma L. Burlingame; Tiegang Lu; Kang Chong; Zhi-Yong Wang

    2010-01-01

    Brassinosteroids (BRs) are essential hormones for growth and development of plant. In rice, BRs regulate multiple developmental processes and affect many important traits such as height, leaf angle, fertility and seed filling. We identified brassinosteroid-regulated proteins in rice using proteomic approaches and performed functional analysis of some BR-regulated proteins by overexpression experiments. Using two-dimensional difference gel electrophoresis (2-D DIGE) followed by protein identification by mass spectrometry, we compared proteomic differences in the shoots and roots of the BR-insensitive mutant d61-4 and BR-deficient mutant brd1-3. We identified a large number of proteins differentially expressed in the mutants compared with wild type control. These include a glycine-rich RNA-binding protein (OsGRP1)and a DREPP2 protein, which showed reduced levels in the BR mutants. Overexpression of these two proteins partially suppressed the dwarf phenotype of the Arabidopsis BR-insensitive mutant bri1-5. In contrast to the reduced protein level, the RNA level of OsGRP1 was not significantly affected in the BR mutants or by BR treatment, suggesting BR regulation of OsGRP1 at the posttranslational level. This study identifies many BR-regulated proteins and demonstrates that OsGRP1 functions downstream in the BR signal transduction pathway to promote cell expansion.

  6. Modifying the DPClus algorithm for identifying protein complexes based on new topological structures

    Directory of Open Access Journals (Sweden)

    Wang Jian-xin

    2008-09-01

    Full Text Available Abstract Background Identification of protein complexes is crucial for understanding principles of cellular organization and functions. As the size of protein-protein interaction set increases, a general trend is to represent the interactions as a network and to develop effective algorithms to detect significant complexes in such networks. Results Based on the study of known complexes in protein networks, this paper proposes a new topological structure for protein complexes, which is a combination of subgraph diameter (or average vertex distance and subgraph density. Following the approach of that of the previously proposed clustering algorithm DPClus which expands clusters starting from seeded vertices, we present a clustering algorithm IPCA based on the new topological structure for identifying complexes in large protein interaction networks. The algorithm IPCA is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm IPCA recalls more known complexes than previously proposed clustering algorithms, including DPClus, CFinder, LCMA, MCODE, RNSC and STM. Conclusion The proposed algorithm based on the new topological structure makes it possible to identify dense subgraphs in protein interaction networks, many of which correspond to known protein complexes. The algorithm is robust to the known high rate of false positives and false negatives in data from high-throughout interaction techniques. The program is available at http://netlab.csu.edu.cn/bioinformatics/limin/IPCA.

  7. 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.

  8. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    Science.gov (United States)

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  9. Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and non-transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Lee Tzong-Yi

    2010-10-01

    Full Text Available Abstract Background While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides from precursors to form intermediate structures. Due to the difficulty of experimental identification, several works have used computational methods to identify glycosylation sites. Results By investigating glycosylated sites that contain various motifs between Transmembrane (TM and non-Transmembrane (non-TM proteins, this work presents a novel method, GlycoRBF, that implements radial basis function (RBF networks with significant amino acid pairs (SAAPs for identifying O-linked glycosylated serine and threonine on TM proteins and non-TM proteins. Additionally, a membrane topology is considered for reducing the false positives on glycosylated TM proteins. Based on an evaluation using five-fold cross-validation, the consideration of a membrane topology can reduce 31.4% of the false positives when identifying O-linked glycosylation sites on TM proteins. Via an independent test, GlycoRBF outperforms previous O-linked glycosylation site prediction schemes. Conclusion A case study of Cyclic AMP-dependent transcription factor ATF-6 alpha was presented to demonstrate the effectiveness of GlycoRBF. Web-based GlycoRBF, which can be accessed at http://GlycoRBF.bioinfo.tw, can identify O-linked glycosylated serine and threonine effectively and efficiently. Moreover, the structural topology of Transmembrane (TM proteins with glycosylation sites is provided to users. The stand-alone version of GlycoRBF is also available for high throughput data analysis.

  10. Novel proteins identified in the insoluble byssal matrix of the freshwater zebra mussel.

    Science.gov (United States)

    Gantayet, Arpita; Rees, David J; Sone, Eli D

    2014-04-01

    The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.

  11. Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

    Science.gov (United States)

    Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

    2013-01-01

    Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

  12. Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

    Science.gov (United States)

    Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

    2013-03-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

  13. A Novel Approach for Identifying the Heme-Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    Xiaolei Li; Rong Wang; Zhongsheng Sun; Zuyuan Xu; Jingyue Bao; Xiuqing Zhang; Xiaoli Feng; Siqi Liu; Xiaoshan Wang; Kang Zhao; Zhengfeng Zhou; Caifeng Zhao; Ren Yan; Liang Lin; Tingting Lei; Jianning Yin

    2003-01-01

    Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of hemebinding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling hemne-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by hemeagarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  14. A Novel Approach for Identifying the Heme—Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    XiaoleiLi; XiaoshanWang; KangZhao; ZhengfengZhou; CaifengZhao; RenYan; LiangLin; TingtingLei; JianningYin; RongWang; ZhongshengSun; ZuyuanXu; JingyueBao; XiugingZhang; XiaoliFeng; SiqiLiu

    2003-01-01

    Heme is a key cofactor in aerobic life,both in eukaryotes and prokaryotes.Because of the high reactivity of ferrous protoporphyrin IX,the reactions of heme in cells are often carried out through heme-protein complexes.Traditionally studies of hemebinding proteins have been approached on a case by case basis,thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues.The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1)purification of heme-binding proteins by hemeagarose,an affinity chromatographic resin;2)isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis;3)identification of heme-binding proteins by mass spectrometry.In five mouse tissues,over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF,in which most proteins belong to heme related.This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  15. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    Science.gov (United States)

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  16. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    Science.gov (United States)

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  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. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva

    Energy Technology Data Exchange (ETDEWEB)

    Mudenda, Lwiindi; Aguilar Pierle, Sebastian; Turse, Joshua E.; Scoles, Glen A.; Purvine, Samuel O.; Nicora, Carrie D.; Clauss, Therese RW; Ueti, Massaro W.; Brown, Wendy C.; Brayton, Kelly A.

    2014-08-07

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

  19. Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

    Science.gov (United States)

    Lu, Yuwen; McGavin, Wendy; Cock, Peter J A; Schnettler, Esther; Yan, Fei; Chen, Jianping; MacFarlane, Stuart

    2015-11-01

    Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8.

  20. SR proteins Asf/SF2 and 9G8 interact to activate enhancer-dependent intron D splicing of bovine growth hormone pre-mRNA in vitro.

    Science.gov (United States)

    Li, X; Shambaugh, M E; Rottman, F M; Bokar, J A

    2000-01-01

    The alternative splicing of the last intron (intron D) of bovine growth hormone (bGH) pre-mRNA requires a down-stream exonic splicing enhancer (FP/ESE). The presence of at least one SR protein has been shown to be essential for FP/ESE function and splicing of intron D in in vitro splicing assays. However, in vitro reconstitution of splicing using individual purified SR proteins may not accurately reflect the true complexity of alternative splicing in an intact nucleus, where multiple SR proteins in varying amounts are likely to be available simultaneously. Here, a panel of recombinant baculovirus-expressed SR proteins was produced and tested for the ability to activate FP/ESE-dependent splicing. Individual recombinant SR proteins differed significantly in their activity in promoting intron D splicing. Among the recombinant SR proteins tested, SRp55 was the most active, SC35 showed very little activity, and ASF/SF2 and 9G8 individually had intermediate activity. At least one SR protein (ASF/SF2) bound to the FP/ESE with characteristics of a cooperative interaction. Most interestingly, low concentrations of ASF/SF2 and 9G8 acted synergistically to activate intron D splicing. This was due in part to synergistic binding to the FP/ESE. Splicing of bGH intron D is inherently complex, and is likely controlled by an interaction of the FP/ESE with several trans-acting protein factors acting both independently and cooperatively. This level of complexity may be required for precise control of alternative splicing by an exon sequence, which simultaneously is constrained to maintain translational integrity of the mature mRNA. PMID:11142383

  1. Identifying proteins in zebrafish embryos using spectral libraries generated from dissected adult organs and tissues.

    Science.gov (United States)

    van der Plas-Duivesteijn, Suzanne J; Mohammed, Yassene; Dalebout, Hans; Meijer, Annemarie; Botermans, Anouk; Hoogendijk, Jordy L; Henneman, Alex A; Deelder, André M; Spaink, Herman P; Palmblad, Magnus

    2014-03-07

    Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14,164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

  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

    of cell walls, starch and proteins. Of these proteins, 35% had not been identified in previous in planta or in vitro studies, 70% were predicted to contain signal peptides and a further 16% may be secreted in a nonclassical manner. Proteins identified in the 72 spots showing differential appearance...... 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...... and wheat spikelets harvested at 2−6 days after inoculation. In addition, a clear difference in the accumulation of fungal biomass and the extent of fungal‐induced proteolysis of plant β‐amylase was observed in barley and wheat. The present study considerably expands the current database of F. graminearum...

  3. Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

    Directory of Open Access Journals (Sweden)

    Gu Jenny

    2007-02-01

    Full Text Available Abstract Background The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2, a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. Results TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2. The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. Conclusion The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function.

  4. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins.

    Directory of Open Access Journals (Sweden)

    Defne Surujon

    Full Text Available The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species or believed to lack (mammals HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms.

  5. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins.

    Science.gov (United States)

    Surujon, Defne; Ratner, David I

    2016-01-01

    The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms.

  6. 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.

  7. A Method to Identify p62's UBA Domain Interacting Proteins

    Directory of Open Access Journals (Sweden)

    Pridgeon Julia W.

    2003-01-01

    Full Text Available The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays.

  8. Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance.

    Science.gov (United States)

    González-Torralva, Fidel; Brown, Adrian P; Chivasa, Stephen

    2017-02-15

    Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed.

  9. Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance

    Science.gov (United States)

    González-Torralva, Fidel; Brown, Adrian P.; Chivasa, Stephen

    2017-01-01

    Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed. PMID:28198407

  10. Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

    Science.gov (United States)

    Lu, Cheng-Tsung; Chen, Shu-An; Bretaña, Neil Arvin; Cheng, Tzu-Hsiu; Lee, Tzong-Yi

    2011-10-01

    In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (http://csb.cse.yzu.edu.tw/Carboxylator/), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.

  11. 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

  12. 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.

  13. 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.

  14. Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

    Directory of Open Access Journals (Sweden)

    Sibut Vonick

    2010-01-01

    Full Text Available Abstract Background As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Results Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference. The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in

  15. PCE-FR: A Novel Method for Identifying Overlapping Protein Complexes in Weighted Protein-Protein Interaction Networks Using Pseudo-Clique Extension Based on Fuzzy Relation.

    Science.gov (United States)

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Ding, Pingjian

    2016-10-01

    Identifying overlapping protein complexes in protein-protein interaction (PPI) networks can provide insight into cellular functional organization and thus elucidate underlying cellular mechanisms. Recently, various algorithms for protein complexes detection have been developed for PPI networks. However, majority of algorithms primarily depend on network topological feature and/or gene expression profile, failing to consider the inherent biological meanings between protein pairs. In this paper, we propose a novel method to detect protein complexes using pseudo-clique extension based on fuzzy relation (PCE-FR). Our algorithm operates in three stages: it first forms the nonoverlapping protein substructure based on fuzzy relation and then expands each substructure by adding neighbor proteins to maximize the cohesive score. Finally, highly overlapped candidate protein complexes are merged to form the final protein complex set. Particularly, our algorithm employs the biological significance hidden in protein pairs to construct edge weight for protein interaction networks. The experiment results show that our method can not only outperform classical algorithms such as CFinder, ClusterONE, CMC, RRW, HC-PIN, and ProRank +, but also achieve ideal overall performance in most of the yeast PPI datasets in terms of composite score consisting of precision, accuracy, and separation. We further apply our method to a human PPI network from the HPRD dataset and demonstrate it is very effective in detecting protein complexes compared to other algorithms.

  16. Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon

    Directory of Open Access Journals (Sweden)

    Schmidt Silvia

    2010-05-01

    Full Text Available Abstract Background Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species. Results Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase, the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases, water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis. Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not. Conclusions We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study

  17. 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.

  18. 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.

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

    NARCIS (Netherlands)

    S. Ligthart (Symen); P.S. de Vries (Paul); A.G. Uitterlinden (André); A. Hofman (Albert); O.H. Franco (Oscar); D.I. Chasman (Daniel); A. Dehghan (Abbas); J. Dupuis (Josée); M. Barbalic (maja); J.C. Bis (Joshua); G. Eiriksdottir (Gudny); Lu, C. (Chen); N. Pellikka (Niina); H. Wallaschofski (Henri); J. Kettunen (Johannes); Henneman, P. (Peter); J. Baumert (Jens); D.P. Strachan (David); C. Fuchsberger (Christian); V. Vitart (Veronique); J.F. Wilson (James F); Paré, G. (Guillaume); S. Naitza (Silvia); M.E. Rudock (Megan); I. Surakka (Ida); E.J.C. de Geus (Eco); B.Z. Alizadeh (Behrooz); J.M. Guralnik (Jack); A.R. Shuldiner (Alan); T. Tanaka (Toshiko); R.Y.L. Zee (Robert); R.B. Schnabel (Renate); V. Nambi (Vijay); M. Kavousi (Maryam); S. Ripatti (Samuli); M. Nauck (Matthias); Smith, N.L. (Nicholas L.); A.V. Smith (Albert Vernon); Sundvall, J. (Jouko); P. Scheet (Paul); Y. Liu (Yongmei); A. Ruokonen (Aimo); L.M. Rose (Lynda); M.G. Larson (Martin); R.C. Hoogeveen (Ron); N.B. Freimer (Nelson); A. Teumer (Alexander); R.P. Tracy (Russell); L.J. Launer (Lenore); J.E. Buring (Julie); J.F. Yamamoto (Jennifer); A.R. Folsom (Aaron); E.J.G. Sijbrands (Eric); J.S. Pankow (James); P. Elliott (Paul); J.F. Keaney (John); Sun, W. (Wei); A.-P. Sarin; M. Fontes (Michel); S. Badola (Sunita); B.C. Astor (Brad); Pouta, A. (Anneli); Werda, K. (Karl); K.H. Greiser (Karin Halina); O. Kuss (Oliver); Schwabedissen, H.E.M.Z. (Henriette E. Meyer Zu); Thiery, J. (Joachim); Y. Jamshidi (Yalda); Nolte, I.M. (Ilja M.); N. Soranzo (Nicole); T.D. Spector (Timothy); H. Völzke (Henry); A.N. Parker (Alex); T. Aspelund (Thor); Bates, D. (David); Young, L. (Lauren); K. Tsui (Kim); D.S. Siscovick (David); X. Guo (Xiuqing); Rotter, J.I. (Jerome I.); M. Uda (Manuela); D. Schlessinger; I. Rudan (Igor); A.A. Hicks (Andrew); B.W.J.H. Penninx (Brenda); B. Thorand (Barbara); C. Gieger (Christian); J. Coresh (Josef); G.A.H.M. Willemsen (Gonneke); T.B. Harris (Tamara); M.-R. Jarvelin (Marjo-Riitta); K.M. Rice (Kenneth); D. Radke (Dörte); V. Salomaa (Veikko); J.A.P. Willems van Dijk (Ko); E.A. Boerwinkle (Eric); R.S. Vasan (Ramachandran Srini); L. Ferrucci (Luigi); Q. Gibson (Quince); S. Bandinelli (Stefania); H. Snieder (Harold); D.I. Boomsma (Dorret); X. Xiao (Xiangjun); H. Campbell (Harry); C. Hayward (Caroline); P.P. Pramstaller (Peter Paul); C.M. van Duijn (Cock); L. Peltonen (Leena Johanna); B.M. Psaty (Bruce); V. Gudnason (Vilmundur); P.M. Ridker (Paul); G. Homuth (Georg); W. Koenig (Wolfgang); C. Ballantyne (Christie); J.C.M. Witteman (Jacqueline); E.J. Benjamin (Emelia); M. Perola (Markus); Chasman., D.I. (Daniel I.)

    2015-01-01

    textabstractPleiotropic 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

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

    NARCIS (Netherlands)

    Ligthart, Symen; de Vries, Paul S.; Uitterlinden, André G.; Hofman, Albert; Franco, Oscar H.; Chasman, Daniel I.; Dehghan, Abbas; Dupuis, Josée; Barbalic, Maja; Bis, Joshua C.; Eiriksdottir, Gudny; Lu, Chen; Pellikka, Niina; Wallaschofski, Henri; Kettunen, Johannes; Henneman, Peter; Baumert, Jens; Strachan, David P.; Fuchsberger, Christian; Vitart, Veronique; Wilson, James F.; Paré, Guillaume; Naitza, Silvia; Rudock, Megan E.; Surakka, Ida; De Geus, Eco J. C.; Alizadeh, Behrooz Z.; Guralnik, Jack M. D.; Shuldiner, Alan; Tanaka, Toshiko; Zee, Robert Y. L.; Schnabel, Renate B.; Nambi, Vijay; Kavousi, Maryam; Ripatti, Samuli; Nauck, Matthias; Smith, Nicholas L.; Smith, Albert V.; Sundvall, Jouko; Scheet, Paul; Liu, Yongmei; Ruokonen, Aimo; Rose, Lynda M.; Larson, Martin G.; Hoogeveen, Ron C.; Freimer, Nelson B.; Teumer, Alexander; Tracy, Russell P.; Launer, Lenore J.; Buring, Julie E.; Yamamoto, Jennifer F.; Folsom, Aaron R.; Sijbrands, Eric J. G.; Pankow, James; Elliott, Paul; Keaney, John F.; Sun, Wei; Sarin, Antti-Pekka; Fontes, João D.; Badola, Sunita; Astor, Brad C.; Pouta, Anneli; Werda, Karl; Greiser, Karin H.; Kuss, Oliver; Schwabedissen, Henriette E. Meyer Zu; Thiery, Joachim; Jamshidi, Yalda; Nolte, Ilja M.; Soranzo, Nicole; Spector, Timothy D.; Völzke, Henry; Parker, Alexander N.; Aspelund, Thor; Bates, David; Young, Lauren; Tsui, Kim; Siscovick, David S.; Guo, Xiuqing; Rotter, Jerome I.; Uda, Manuela; Schlessinger, David; Rudan, Igor; Hicks, Andrew A.; Penninx, Brenda W.; Thorand, Barbara; Gieger, Christian; Coresh, Joe; Willemsen, Gonneke; Harris, Tamara B.; Järvelin, Marjo-Riitta; Rice, Kenneth; Radke, Dörte; Salomaa, Veikko; Van Dijk, Ko Willems; Boerwinkle, Eric; Vasan, Ramachandran S.; Ferrucci, Luigi; Gibson, Quince D.; Bandinelli, Stefania; Snieder, Harold; Boomsma, Dorret I.; Xiao, Xiangjun; Campbell, Harry; Hayward, Caroline; Pramstaller, Peter P.; Duijn, Cornelia Mvan; Peltonen, Leena; Psaty, Bruce M.; Gudnason, Vilmundur; Ridker, Paul M.; Homuth, Georg; Koenig, Wolfgang; Ballantyne, Christie M.; Witteman, Jacqueline C. M.; Benjamin, Emelia J.; Perola, Markus; Chasman., Daniel I.

    2015-01-01

    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 back

  1. Unique motifs identify PIG-A proteins from glycosyltransferases of the GT4 family

    Directory of Open Access Journals (Sweden)

    Bhattacharya Alok

    2008-06-01

    Full Text Available Abstract Background The first step of GPI anchor biosynthesis is catalyzed by PIG-A, an enzyme that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol. This protein is present in all eukaryotic organisms ranging from protozoa to higher mammals, as part of a larger complex of five to six 'accessory' proteins whose individual roles in the glycosyltransferase reaction are as yet unclear. The PIG-A gene has been shown to be an essential gene in various eukaryotes. In humans, mutations in the protein have been associated with paroxysomal noctural hemoglobuinuria. The corresponding PIG-A gene has also been recently identified in the genome of many archaeabacteria although genes of the accessory proteins have not been discovered in them. The present study explores the evolution of PIG-A and the phylogenetic relationship between this protein and other glycosyltransferases. Results In this paper we show that out of the twelve conserved motifs identified by us eleven are exclusively present in PIG-A and, therefore, can be used as markers to identify PIG-A from newly sequenced genomes. Three of these motifs are absent in the primitive eukaryote, G. lamblia. Sequence analyses show that seven of these conserved motifs are present in prokaryote and archaeal counterparts in rudimentary forms and can be used to differentiate PIG-A proteins from glycosyltransferases. Using partial least square regression analysis and data involving presence or absence of motifs in a range of PIG-A and glycosyltransferases we show that (i PIG-A may have evolved from prokaryotic glycosyltransferases and lipopolysaccharide synthases, members of the GT4 family of glycosyltransferases and (ii it is possible to uniquely classify PIG-A proteins versus glycosyltransferases. Conclusion Besides identifying unique motifs and showing that PIG-A protein from G. lamblia and some putative PIG-A proteins from archaebacteria are evolutionarily closer to

  2. 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.

  3. An ensemble method with hybrid features to identify extracellular matrix proteins.

    Science.gov (United States)

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc.

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

    Science.gov (United States)

    Chan, Yuk-Kit; Zhang, Huoming; Liu, Pei; Tsao, Sai-Wah; Lung, Maria Li; Mak, Nai-Ki; Ngok-Shun Wong, Ricky; Ying-Kit Yue, Patrick

    2015-10-15

    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.

  5. FunMod: A Cytoscape Plugin for Identifying Functional Modules in Undirected Protein–Protein Networks

    Directory of Open Access Journals (Sweden)

    Massimo Natale

    2014-08-01

    Full Text Available The characterization of the interacting behaviors of complex biological systems is a primary objective in protein–protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein–protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies.

  6. Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.

    Science.gov (United States)

    Yang, Jun; Fuller, Peter J; Morgan, James; Shibata, Hirotaka; McDonnell, Donald P; Clyne, Colin D; Young, Morag J

    2014-09-01

    The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.

  7. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets.

    Science.gov (United States)

    Vinayagam, Arunachalam; Gibson, Travis E; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-05-03

    The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets.

  8. High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

    Science.gov (United States)

    Gu, Minghao; Kilduff, James E; Belfort, Georges

    2012-02-01

    Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).

  9. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  10. Suppression of a defect in mitochondrial protein import identifies cytosolic proteins required for viability of yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Dunn, Cory D; Jensen, Robert E

    2003-01-01

    The TIM22 complex, required for the insertion of imported polytopic proteins into the mitochondrial inner membrane, contains the nonessential Tim18p subunit. To learn more about the function of Tim18p, we screened for high-copy suppressors of the inability of tim18Delta mutants to live without mitochondrial DNA (mtDNA). We identified several genes encoding cytosolic proteins, including CCT6, SSB1, ICY1, TIP41, and PBP1, which, when overproduced, rescue the mtDNA dependence of tim18Delta cells. Furthermore, these same plasmids rescue the petite-negative phenotype of cells lacking other components of the mitochondrial protein import machinery. Strikingly, disruption of the genes identified by the different suppressors produces cells that are unable to grow without mtDNA. We speculate that loss of mtDNA leads to a lowered inner membrane potential, and subtle changes in import efficiency can no longer be tolerated. Our results suggest that increased amounts of Cct6p, Ssb1p, Icy1p, Tip41p, and Pbp1p help overcome the problems resulting from a defect in protein import. PMID:14504216

  11. 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.

  12. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    Directory of Open Access Journals (Sweden)

    Chunxiao Yu

    Full Text Available The Ferric uptake regulatory protein (Fur is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS, to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  13. 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...

  14. 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

    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...... to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could...

  15. 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...... 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...

  16. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    Directory of Open Access Journals (Sweden)

    Stephen R Spindler

    Full Text Available Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR, platelet-derived growth factor (PDGF/vascular endothelial growth factor (VEGF receptors, G-protein coupled receptor (GPCR, Janus kinase (JAK/signal transducer and activator of transcription (STAT, the insulin and insulin-like growth factor (IGFI receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38, c-Jun N-terminal kinase (JNK and protein kinase C (PKC. If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  17. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    Science.gov (United States)

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

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

    Science.gov (United States)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    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 are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive 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, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  19. 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...

  20. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

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    Akiyoshi Uezumi

    2016-08-01

    Full Text Available Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

  1. Novel snail1 target proteins in human colon cancer identified by proteomic analysis.

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    María Jesús Larriba

    Full Text Available BACKGROUND: The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT, a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT. METHODOLOGY/PRINCIPAL FINDINGS: We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4 that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6 and the Splicing factor proline/glutamine-rich (SFPQ, was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3epsilon, 14-3-3tau, 14-3-3zeta and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3sigma and Snail1 expression in human colorectal tumors. CONCLUSIONS/SIGNIFICANCE: We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.

  2. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison

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    Wang Yong

    2006-09-01

    Full Text Available Abstract Background Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. Results We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs can be identified by our method. The software SAMO is available upon request from the authors, or from http://zhangroup.aporc.org/bioinfo/samo/ and http://intelligent.eic.osaka-sandai.ac.jp/chenen/samo.htm. Conclusion A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven.

  3. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

    Science.gov (United States)

    Agrawal, Parul; Hardin, Paul E

    2016-12-07

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  4. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock

    Directory of Open Access Journals (Sweden)

    Parul Agrawal

    2016-12-01

    Full Text Available Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC complexes activate transcription of period (per and timeless (tim genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Edwin Lasonder

    2008-10-01

    Full Text Available 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, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins 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 be essential for sporozoite infectivity to humans.

  7. A proteomic approach to identify proteins from Trichuris trichiura extract with immunomodulatory effects.

    Science.gov (United States)

    Santos, L N; Gallo, M B C; Silva, E S; Figueiredo, C A V; Cooper, P J; Barreto, M L; Loureiro, S; Pontes-de-Carvalho, L C; Alcantara-Neves, N M

    2013-01-01

    Infections with Trichuris trichiura and other trichurid nematodes have been reported to display protective effects against atopy, allergic and autoimmune diseases. The aims of the present study were to investigate the immunomodulatory properties of T. trichiura adult worm extract (TtE) and its fractions (TtEFs) on the production of cytokines by peripheral blood mononuclear cells and to identify their proteinaceous components. Fourteen TtEFs were obtained by ion exchange chromatography and tested for effects on cytokine production by peripheral blood mononuclear cells. The molecular constituents of the six most active fractions were evaluated using nano-LC/mass spectrometry. The homology between T. trichiura and the related nematode Trichinella spiralis was used to identify 12 proteins in TtEFs. Among those identified, fructose biphosphate aldolase, a homologue of macrophage migration inhibitory factor and heat-shock protein 70 may contribute to the immunomodulatory effects of TtEFs. The identification of such proteins could lead to the development of novel drugs for the therapy of allergic and other inflammatory diseases.

  8. Erosion of the Alps: use of Rb-Sr isotopic data from molassic sediments to identify the ages of the metamorphism recorded by the eroded rocks; Erosion des Alpes: histoire metamorphique des roches erodees par l`analyse du couple Rb-Sr des sediments molassiques

    Energy Technology Data Exchange (ETDEWEB)

    Henry, P.; Deloule, E. [Centre National de la Recherche Scientifique (CNRS), 54 - Nancy (France). Centre de Recherches Petrographiques et Geochimiques; Michard, A. [Aix-Marseille-3 Univ., 13 - Marseille (France)

    1994-06-01

    Rb-Sr isotopic data from Oligocene and Miocene peri-alpine molassic sediments allow us to identify the different periods for which the eroded rocks have or have not recorded an alpine metamorphism. The Chattian and the Burdigalian sediments result from the erosion of rocks for which the latest metamorphic event was variscan, while the Stampian, Aquitanian and ``Helvetian`` sediments show evidence for the erosion of rocks which have recorded alpine metamorphic events. The application of this method to old detrital sediments could permit determination of the ages of the tectonic events which occurred in the sediment source regions. (authors). 18 refs., 6 figs.

  9. 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

    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...

  10. 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

  11. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    Science.gov (United States)

    Achkar, Jacqueline M.; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O.; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  12. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Achkar

    2015-09-01

    Full Text Available Biomarkers for active tuberculosis (TB are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI, uninfected states, or respiratory diseases other than TB (ORD. Serum samples from 209 HIV uninfected (HIV− and co-infected (HIV+ individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1, or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6, respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB. These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

  13. 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

    2016-01-01

    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

  14. 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

  15. 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

  16. The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola.

    Science.gov (United States)

    Krijger, Jorrit-Jan; Horbach, Ralf; Behr, Michael; Schweizer, Patrick; Deising, Holger B; Wirsel, Stefan G R

    2008-10-01

    The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

  17. 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

  18. Effect of potassium iodate and potassium iodide on the SR-B1 protein expression of abdominal aorta in rats%碘酸钾和碘化钾对大鼠腹主动脉SR-B1蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    曹晓晓; 徐菁; 谷云有; 王海燕; 李秀维; 王建强; 刘列钧

    2013-01-01

    目的 观察不同剂量的碘酸钾和碘化钾对大鼠腹主动脉SR-B1蛋白表达的影响.方法 根据喂养碘剂和剂量不同将Wistar大鼠随机分为适碘组(KI、KIO3),10倍高碘组(10KI、10KIO3)、50倍高碘组(50KI、50KIO3),100倍高碘组(100KI、100KIO3).喂养半年后,利用免疫蛋白印迹分析(Western-blot)检测大鼠腹主动脉中SR-B1蛋白表达水平.结果 大鼠腹主动脉重量无组间统计学差异(P>0.05);大鼠动脉SR-B1蛋白水平在适碘组之间无差异(P>0.05),而在高剂量的碘酸钾组中,SR-B1蛋白水平均低于等剂量的碘化钾组(P<0.05);SR-B1的蛋白表达水平随着碘酸钾喂养剂量的增加而下降,但在不同剂量碘化钾组中无明显变化趋势.结论 与碘化钾相比,高剂量的碘酸钾抑制了大鼠动脉SR-B1蛋白表达水平.%Objective To observe the effect of potassium iodate and potassium iodide on the SR - Bl protein expression of abdominal aorta in rats. Methods The SPF wistar rats were randomly divided into 8 groups, normal iodide groups ( KI, KIO3), 10 fold iodide groups (10KI, 10KIO3), 50 fold iodide groups (50KI, 50KIO3) and 100 fold iodide groups (100KI , 100KIO3). After 6 months treatment, the SR - Bl protein levels in abdominal aorta were determined by western - blot analysis. Results The weights of rat abdominal aorta showed no statistically significant change among 8 groups ( P >0. 05); The SR - Bl protein expression displayed no remarkable difference between the group of KI and KIO3( P >0.05). But the protein level of SR - Bl in high dose groups of potassium iodate (10KIO3, 50KIO3, and 100KIO3) were higher than those in isodose groups of potassium iodide (10KI, 50KI and 100KI) ( P <0. 05); The SR - Bl protein level decreased with the increase of potassium iodate feeding dosage, but there was no apparent trend among the groups of potassium iodide. Conclusion Compared with potassium iodide, high dose potassium iodate can depress the SR - Bl protein

  19. Extracellular proteome of Trichoderma harzianum to identify proteins with biotechnological value

    Institute of Scientific and Technical Information of China (English)

    Ambrosino P; Lorito M; Scala V; Marra R; Vinale F; Soriente I; Ferraioli S; Carbone V; Ruocco M; Woo S L

    2004-01-01

    @@ Trichoderma harzianum strain T22 parasitizes and controls many phytopatogenic fungi and is applied commercially as biological control agent. The production of hydrolitic enzymes appears to be a key factor in the parasitic process. We tested the endo-esochitinolitic and glucanolitic activities of culture filtrates of T22 grown under carbon and nitrogen starvation or in presence of biomass or cell walls of the phytopathogenic fungi Botrytis cinerea , Rhizoctonia solani and Pythium ultimum. The highest level of enzimatic activities was found in culture where the mycoparasite interacted with a phytopathogenic fungus. Therefore we used a proteomic approach to investigate changes in the complex mixture of extracellular proteins secreted by T. harzianum strain T22 in order to identify proteins of potential biotechnology value for commercial and industrial use. Proteome technology has greatly enhanced our ability to conduct functional genomics studies. Nevertheless only a few studies have been published so far on the fungal extracellular proteome. Sample preparation remains the most critical step in analyses based on two-dimensional gel electrophoresis (2-DE), and it requires to be optimized for each specific application. In this study, our first aim was to set up the extraction protocol of the extracellular proteins secreted by T . harzianum strain T22 when it was grown in vitro . The secreted proteins were analysed by two-dimensional electrophoresis (2-DE) and substantial changes in the extracellular proteome of the mycoparasite have been observed. Comparing the 2D maps of the fungus grown in minimal medium with glycerol as carbon source (used as control condition) with those obtained in inducing conditions, a lot of novel proteins appeared. The higher number of novel and upregulated spots was obtained in the presence of Rhizoctonia solani biomass. Other spots were specifically up-regulated by the interaction with different plant pathogens. Differentially expressed

  20. 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.

  1. A phenotypic assay to identify Chikungunya virus inhibitors targeting the nonstructural protein nsP2.

    Science.gov (United States)

    Lucas-Hourani, Marianne; Lupan, Alexandru; Desprès, Philippe; Thoret, Sylviane; Pamlard, Olivier; Dubois, Joëlle; Guillou, Catherine; Tangy, Frédéric; Vidalain, Pierre-Olivier; Munier-Lehmann, Hélène

    2013-02-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

  2. Using phylogenomic patterns and gene ontology to identify proteins of importance in plant evolution.

    Science.gov (United States)

    Cibrián-Jaramillo, Angélica; De la Torre-Bárcena, Jose E; Lee, Ernest K; Katari, Manpreet S; Little, Damon P; Stevenson, Dennis W; Martienssen, Rob; Coruzzi, Gloria M; DeSalle, Rob

    2010-07-12

    We use measures of congruence on a combined expressed sequenced tag genome phylogeny to identify proteins that have potential significance in the evolution of seed plants. Relevant proteins are identified based on the direction of partitioned branch and hidden support on the hypothesis obtained on a 16-species tree, constructed from 2,557 concatenated orthologous genes. We provide a general method for detecting genes or groups of genes that may be under selection in directions that are in agreement with the phylogenetic pattern. Gene partitioning methods and estimates of the degree and direction of support of individual gene partitions to the overall data set are used. Using this approach, we correlate positive branch support of specific genes for key branches in the seed plant phylogeny. In addition to basic metabolic functions, such as photosynthesis or hormones, genes involved in posttranscriptional regulation by small RNAs were significantly overrepresented in key nodes of the phylogeny of seed plants. Two genes in our matrix are of critical importance as they are involved in RNA-dependent regulation, essential during embryo and leaf development. These are Argonaute and the RNA-dependent RNA polymerase 6 found to be overrepresented in the angiosperm clade. We use these genes as examples of our phylogenomics approach and show that identifying partitions or genes in this way provides a platform to explain some of the more interesting organismal differences among species, and in particular, in the evolution of plants.

  3. Virtual screening studies to identify novel inhibitors for Sigma F protein of Mycobacterium tuberculosis.

    Science.gov (United States)

    Mustyala, Kiran Kumar; Malkhed, Vasavi; Chittireddy, Venkataramana Reddy; Vuruputuri, Uma

    2015-12-01

    Tuberculosis (TB) is one of the oldest threats to public health. TB is caused by the pathogen Mycobacterium tuberculosis (MTB). The Sigma factors are essential for the survival of MTB. The Sigma factor Sigma F (SigF) regulates genes expression under stress conditions. The SigF binds to RNA polymerase and forms a holoenzyme, which initiates the transcription of various genes. The Usfx, an anti-SigF protein, binds to SigF and alters the transcription initiation and gene expression. In the present work, virtual screening studies are taken up to identify the interactions between SigF and small molecular inhibitors which can inhibit the formation of holoenzyme. The studies reveal that ARG 104 and ARG 224 amino acid residues of SigF protein are forming important binding interactions with the ligands. The in silico ADME properties for the ligand data set are calculated to check the druggability of the molecules.

  4. Origins of invasive piscivores determined from the strontium isotope ratio (87Sr/86Sr) of otoliths

    Science.gov (United States)

    Wolff, Brian A.; Johnson, Brett M.; Breton, Andre R.; Martinez, Patrick J.; Winkelman, Dana L.; Gillanders, Bronwyn

    2012-01-01

    We examined strontium isotope ratios (87Sr/86Sr) in fish otoliths to determine the origins of invasive piscivores in the Upper Colorado River Basin (UCRB, western USA). We examined 87Sr/86Sr from fishes in different reservoirs, as well as the temporal stability and interspecies variability of 87Sr/86Sr of fishes within reservoirs, determined if 87Sr/86Sr would be useful for "fingerprinting" reservoirs where invasive piscivores may have been escaping into riverine habitat of endangered fishes in the UCRB, and looked for evidence that such movement was occurring. Our results showed that in most cases 87Sr/86Sr was unique among reservoirs, overlapped among species in a given reservoir, and was temporally stable across years. We identified the likely reservoir of origin of river-caught fish in some cases, and we were also able to determine the year of possible escapement. The approach allowed us to precisely describe the 87Sr/86Sr fingerprint of reservoir fishes, trace likely origins of immigrant river fish, and exclude potential sources, enabling managers to focus control efforts more efficiently. Our results demonstrate the potential utility of 87Sr/86Sr as a site-specific and temporally stable marker for reservoir fish and its promise for tracking fish movements of invasive fishes in river-reservoir systems.

  5. 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.

  6. High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions.

    Science.gov (United States)

    Hua, Yun; Strock, Christopher J; Johnston, Paul A

    2015-01-01

    This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs. The N-terminus of hDM2 encodes the domain for binding to the transactivating domain of p53, and is expressed as a RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells co-infected with both adenovirus constructs, the binding interactions between hDM2 and p53 result in both biosensors becoming co-localized within the nucleolus. Upon disruption of the p53-hDM2 PPIs, the p53-GFP biosensor remains in the nucleolus while the shuttling hDM2-RFP biosensor redistributes into the cytoplasm. p53-hDM2 PPIs are measured by acquiring fluorescent images of cells co-infected with both adenovirus biosensors on an automated HCS imaging platform and using an image analysis algorithm to quantify the relative distribution of the hDM2-RFP shuttling component of the biosensor between the cytoplasm and nuclear regions of compound treated cells.

  7. 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.

  8. Beta atomic contacts: identifying critical specific contacts in protein binding interfaces.

    Science.gov (United States)

    Liu, Qian; Kwoh, Chee Keong; Hoi, Steven C H

    2013-01-01

    Specific binding between proteins plays a crucial role in molecular functions and biological processes. Protein binding interfaces and their atomic contacts are typically defined by simple criteria, such as distance-based definitions that only use some threshold of spatial distance in previous studies. These definitions neglect the nearby atomic organization of contact atoms, and thus detect predominant contacts which are interrupted by other atoms. It is questionable whether such kinds of interrupted contacts are as important as other contacts in protein binding. To tackle this challenge, we propose a new definition called beta (β) atomic contacts. Our definition, founded on the β-skeletons in computational geometry, requires that there is no other atom in the contact spheres defined by two contact atoms; this sphere is similar to the van der Waals spheres of atoms. The statistical analysis on a large dataset shows that β contacts are only a small fraction of conventional distance-based contacts. To empirically quantify the importance of β contacts, we design βACV, an SVM classifier with β contacts as input, to classify homodimers from crystal packing. We found that our βACV is able to achieve the state-of-the-art classification performance superior to SVM classifiers with distance-based contacts as input. Our βACV also outperforms several existing methods when being evaluated on several datasets in previous works. The promising empirical performance suggests that β contacts can truly identify critical specific contacts in protein binding interfaces. β contacts thus provide a new model for more precise description of atomic organization in protein quaternary structures than distance-based contacts.

  9. Beta atomic contacts: identifying critical specific contacts in protein binding interfaces.

    Directory of Open Access Journals (Sweden)

    Qian Liu

    Full Text Available Specific binding between proteins plays a crucial role in molecular functions and biological processes. Protein binding interfaces and their atomic contacts are typically defined by simple criteria, such as distance-based definitions that only use some threshold of spatial distance in previous studies. These definitions neglect the nearby atomic organization of contact atoms, and thus detect predominant contacts which are interrupted by other atoms. It is questionable whether such kinds of interrupted contacts are as important as other contacts in protein binding. To tackle this challenge, we propose a new definition called beta (β atomic contacts. Our definition, founded on the β-skeletons in computational geometry, requires that there is no other atom in the contact spheres defined by two contact atoms; this sphere is similar to the van der Waals spheres of atoms. The statistical analysis on a large dataset shows that β contacts are only a small fraction of conventional distance-based contacts. To empirically quantify the importance of β contacts, we design βACV, an SVM classifier with β contacts as input, to classify homodimers from crystal packing. We found that our βACV is able to achieve the state-of-the-art classification performance superior to SVM classifiers with distance-based contacts as input. Our βACV also outperforms several existing methods when being evaluated on several datasets in previous works. The promising empirical performance suggests that β contacts can truly identify critical specific contacts in protein binding interfaces. β contacts thus provide a new model for more precise description of atomic organization in protein quaternary structures than distance-based contacts.

  10. Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas.

    Science.gov (United States)

    Wu, Chih-Ching; Hsu, Chia-Wei; Chen, Chi-De; Yu, Chia-Jung; Chang, Kai-Ping; Tai, Dar-In; Liu, Hao-Ping; Su, Wen-Hui; Chang, Yu-Sun; Yu, Jau-Song

    2010-06-01

    Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6-137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.

  11. Protein C/S ratio, an accurate and simple tool to identify carriers of a protein C gene mutation

    NARCIS (Netherlands)

    Libourel, EJ; Meinardi, [No Value; de Kam, PJ; Ruiters, MHJ; van der Meer, J; van der Schaaf, W; Veenstra, R.

    Hereditary protein C deficiency is demonstrated by lowered protein C plasma levels in a patient and at least one first-degree relative. This approach is insufficient in some cases owing to overlapping protein C levels in carriers and non-carriers of a protein C gene mutation. The protein C/S ratio

  12. Endogenous ribosomal protein L29 (RPL29: a newly identified regulator of angiogenesis in mice

    Directory of Open Access Journals (Sweden)

    Dylan T. Jones

    2013-01-01

    Cellular ribosomal protein L29 (RPL29 is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking β3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvβ3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in β3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.

  13. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Directory of Open Access Journals (Sweden)

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  14. A fast iterative-clique percolation method for identifying functional modules in protein interaction networks

    Institute of Scientific and Technical Information of China (English)

    Penggang SUN; Lin GAO

    2009-01-01

    Accumulating evidence suggests that biological systems are composed of interacting, separable, functional modules-groups of vertices within which connections are dense but between which they are sparse. Identifying these modules is likely through capturing the biologically mean-ingful interactions. In recent years, many algorithms have been developed for detecting such structures. These al-gorithms, however, are computationally demanding, which limits their applications. In this paper, we propose a fast iterative-clique percolation method (ICPM) for identifying overlapping functional modules in protein-protein interac-tion (PPI) networks. Our method is based on clique percola-tion method (CPM), and it not only considers the degree of nodes to minimize the search space (the vertices in k-cliques must have the degree of k - 1 at least), but also converts k-cliques to (k - 1)-cliques. It finds k-cliques by append-ing one node to (k - 1)-cliques. By testing our method on PPI networks, our analysis of the yeast PPI network suggeststhat most of these modules have well-supported biological significance.

  15. Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases.

    Science.gov (United States)

    Cui, Danica S; Beaumont, Victor; Ginther, Patrick S; Lipchock, James M; Loria, J Patrick

    2017-07-21

    Drug-like molecules targeting allosteric sites in proteins are of great therapeutic interest; however, identification of potential sites is not trivial. A straightforward approach to identify hidden allosteric sites is demonstrated in protein tyrosine phosphatases (PTP) by creation of single alanine mutations in the catalytic acid loop of PTP1B and VHR. This approach relies on the reciprocal interactions between an allosteric site and its coupled orthosteric site. The resulting NMR chemical shift perturbations (CSPs) of each mutant reveal clusters of distal residues affected by acid loop mutation. In PTP1B and VHR, two new allosteric clusters were identified in each enzyme. Mutations in these allosteric clusters altered phosphatase activity with changes in kcat/KM ranging from 30% to nearly 100-fold. This work outlines a simple method for identification of new allosteric sites in PTP, and given the basis of this method in thermodynamics, it is expected to be generally useful in other systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Identifying G-protein Coupled Receptors Using Weighted Levenshtein Distance and Nearest Neighbor Method

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Xu

    2005-01-01

    G-protein coupled receptors (GPCRs) are a class of seven-helix transmembrane proteins that have been used in bioinformatics as the targets to facilitate drug discovery for human diseases. Although thousands of GPCR sequences have been collected, the ligand specificity of many GPCRs is still unknown and only one crystal structure of the rhodopsin-like family has been solved. Therefore, identifying GPCR types only from sequence data has become an important research issue. In this study, a novel technique for identifying GPCR types based on the weighted Levenshtein distance between two receptor sequences and the nearest neighbor method (NNM) is introduced, which can deal with receptor sequences with different lengths directly. In our experiments for classifying four classes(acetylcholine, adrenoceptor, dopamine, and serotonin) of the rhodopsin-like family of GPCRs, the error rates from the leave-one-out procedure and the leave-half-out procedure were 0.62% and 1.24%, respectively. These results are prior to those of the covariant discriminant algorithm, the support vector machine method, and the NNM with Euclidean distance.

  17. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

    Directory of Open Access Journals (Sweden)

    Marlène Dreux

    2009-02-01

    Full Text Available HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI, a major receptor of high-density lipoprotein (HDL, the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

  18. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

    Directory of Open Access Journals (Sweden)

    Marlène Dreux

    2009-02-01

    Full Text Available HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI, a major receptor of high-density lipoprotein (HDL, the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

  19. 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

  20. 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

  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. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    Science.gov (United States)

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

  3. Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity.

    Science.gov (United States)

    McGarry, David J; Chakravarty, Probir; Wolf, C Roland; Henderson, Colin J

    2015-11-01

    Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography-tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2(-/-) mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2(-/-) mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2(-/-) mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity.

  4. The use of functional chemical-protein associations to identify multi-pathway renoprotectants.

    Directory of Open Access Journals (Sweden)

    Jia Xu

    Full Text Available Typically, most nephropathies can be categorized as complex human diseases in which the cumulative effect of multiple minor genes, combined with environmental and lifestyle factors, determines the disease phenotype. Thus, multi-target drugs would be more likely to facilitate comprehensive renoprotection than single-target agents. In this study, functional chemical-protein association analysis was performed to retrieve multi-target drugs of high pathway wideness from the STITCH 3.1 database. Pathway wideness of a drug evaluated the efficiency of regulation of Kyoto Encyclopedia of Genes and Genomes (KEGG pathways in quantity. We identified nine experimentally validated renoprotectants that exerted remarkable impact on KEGG pathways by targeting a limited number of proteins. We selected curcumin as an illustrative compound to display the advantage of multi-pathway drugs on renoprotection. We compared curcumin with hemin, an agonist of heme oxygenase-1 (HO-1, which significantly affects only one KEGG pathway, porphyrin and chlorophyll metabolism (adjusted p = 1.5×10-5. At the same concentration (10 µM, both curcumin and hemin equivalently mitigated oxidative stress in H2O2-treated glomerular mesangial cells. The benefit of using hemin was derived from its agonistic effect on HO-1, providing relief from oxidative stress. Selective inhibition of HO-1 completely blocked the action of hemin but not that of curcumin, suggesting simultaneous multi-pathway intervention by curcumin. Curcumin also increased cellular autophagy levels, enhancing its protective effect; however, hemin had no effects. Based on the fact that the dysregulation of multiple pathways is implicated in the etiology of complex diseases, we proposed a feasible method for identifying multi-pathway drugs from compounds with validated targets. Our efforts will help identify multi-pathway agents capable of providing comprehensive protection against renal injuries.

  5. Evolutionary rate covariation identifies new members of a protein network required for Drosophila melanogaster female post-mating responses.

    Directory of Open Access Journals (Sweden)

    Geoffrey D Findlay

    2014-01-01

    Full Text Available Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.

  6. A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.

    Science.gov (United States)

    Arnaiz, Blanca; Madrigal-Estebas, Laura; Todryk, Stephen; James, Tharappel C; Doherty, Derek G; Bond, Ursula

    2006-04-08

    The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-gamma (IFN-gamma) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.

  7. Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system.

    Science.gov (United States)

    Petit-Härtlein, Isabelle; Rome, Kevin; de Rosny, Eve; Molton, Florian; Duboc, Carole; Gueguen, Erwan; Rodrigue, Agnès; Covès, Jacques

    2015-12-01

    The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.

  8. Identifying disease-specific genes based on their topological significance in protein networks

    Directory of Open Access Journals (Sweden)

    Cherba David

    2009-03-01

    Full Text Available Abstract Background The identification of key target nodes within complex molecular networks remains a common objective in scientific research. The results of pathway analyses are usually sets of fairly complex networks or functional processes that are deemed relevant to the condition represented by the molecular profile. To be useful in a research or clinical laboratory, the results need to be translated to the level of testable hypotheses about individual genes and proteins within the condition of interest. Results In this paper we describe novel computational methodology capable of predicting key regulatory genes and proteins in disease- and condition-specific biological networks. The algorithm builds shortest path network connecting condition-specific genes (e.g. differentially expressed genes using global database of protein interactions from MetaCore. We evaluate the number of all paths traversing each node in the shortest path network in relation to the total number of paths going via the same node in the global network. Using these numbers and the relative size of the initial data set, we determine the statistical significance of the network connectivity provided through each node. We applied this method to gene expression data from psoriasis patients and identified many confirmed biological targets of psoriasis and suggested several new targets. Using predicted regulatory nodes we were able to reconstruct disease pathways that are in excellent agreement with the current knowledge on the pathogenesis of psoriasis. Conclusion The systematic and automated approach described in this paper is readily applicable to uncovering high-quality therapeutic targets, and holds great promise for developing network-based combinational treatment strategies for a wide range of diseases.

  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. Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

    Directory of Open Access Journals (Sweden)

    Deyi Xiao

    Full Text Available BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs, and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

  11. Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization.

    Science.gov (United States)

    Twigg, Stephen R F; Lloyd, Deborah; Jenkins, Dagan; Elçioglu, Nursel E; Cooper, Christopher D O; Al-Sannaa, Nouriya; Annagür, Ali; Gillessen-Kaesbach, Gabriele; Hüning, Irina; Knight, Samantha J L; Goodship, Judith A; Keavney, Bernard D; Beales, Philip L; Gileadi, Opher; McGowan, Simon J; Wilkie, Andrew O M

    2012-11-02

    Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.

  12. Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein.

    Science.gov (United States)

    Calvert, Amanda E; Kalantarov, Gavreel F; Chang, Gwong-Jen J; Trakht, Ilya; Blair, Carol D; Roehrig, John T

    2011-02-05

    Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.

  13. In vivo cross-linking followed by polyA enrichment to identify yeast mRNA binding proteins.

    Science.gov (United States)

    Mitchell, Sarah F; Parker, Roy

    2015-01-01

    mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand this process, it is necessary to identify the complete set of mRNA binding proteins. This work describes a method for the systematic identification of yeast mRNA binding proteins. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.

  14. Comprehensive antigen screening identifies Moraxella catarrhalis proteins that induce protection in a mouse pulmonary clearance model.

    Directory of Open Access Journals (Sweden)

    Margarita Smidt

    Full Text Available Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22, which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis.

  15. 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.

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

    Science.gov (United States)

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B; Sýkorová, Eva

    2015-01-01

    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 an 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.

  17. Proteome-wide screens for small ubiquitin-like modifier (SUMO) substrates identify Arabidopsis proteins implicated in diverse biological processes

    National Research Council Canada - National Science Library

    Nabil Elrouby; George Coupland

    2010-01-01

    .... Despite this, little is known about SUMO targets in plants. Here we identified 238 Arabidopsis proteins as potential SUMO substrates because they interacted with SUMO-conjugating enzyme and/or SUMO protease (ESD4...

  18. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    OpenAIRE

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M.; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among th...

  19. 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....

  20. 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...

  1. Tear film proteins deposited on high water content contact lenses identified with two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Nielsen, Kim; Vorum, Henrik; Ehlers, Niels; Aagaard, Nicolaj; Hjortdal, Jesper; Honoré, Bent

    2015-11-01

    Tear film proteins adhere to the surface of contact lenses (CLs). While the proteins in the tears have been extensively studied with various proteomic techniques, adhered proteins to CLs are less studied. In this pilot study, we have separated proteins with 2D gel electrophoresis prior to the conventional mass spectrometry (MS) in order to analyse the deposited proteins on hydrogel CLs from myopic patients. pHEMA and PVA hydrogel CLs worn by 3 patients for different time lengths were analysed. After wear, the CLs were frozen at -20°C. Proteins were extracted in lysis buffer, separated on 12% polyacrylamide gels and silver-stained. Protein spots were excised and identified with liquid chromatography - tandem MS. Deposited proteins were extracted with a yield of 26-66 μg and separated by 2D gel electrophoresis. The silver-stained gels showed similar protein patterns independent of the patient, hydrogel type and wear time. Seventy-two spots were analysed with MS, representing at least 12 different tear film proteins or protein fragments. Deposited tear film proteins from a single set of CLs worn for 1 day can successfully be analysed first with 2D gel electrophoresis and subsequently with MS, thus making examination of individual patients possible. The protein composition appeared homogeneous between the test persons which is a necessity for additional comparison analysis. The molecular masses of the identified proteins indicate that protein degradation occurs only as a minor event. Myopic patients were investigated in this pilot study, but the combined techniques can easily be applied to other eye diseases. © 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  2. 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...

  3. Identifying recommended dietary allowances for protein and amino acids: a critique of the 2007 WHO/FAO/UNU report.

    Science.gov (United States)

    Millward, D Joe

    2012-08-01

    The WHO/FAO/UNU (2007) report examines dietary protein and amino acid requirements for all age groups, protein requirements during pregnancy, lactation and catch-up growth in children, the implications of these requirements for developing countries and protein quality evaluation. Requirements were defined as the minimum dietary intake which satisfies the metabolic demand and achieves nitrogen equilibrium and maintenance of the body protein mass, plus the needs for growth in children and pregnancy and lactation in healthy women. Insufficient evidence was identified to enable recommendations for specific health outcomes. A meta analysis of nitrogen balance studies identifies protein requirements for adults 10 % higher than previous values with no influence of gender or age, consistent with a subsequently published comprehensive study. A new factorial model for infants and children, validated on the basis of the adequacy of breast milk protein intakes and involving a lower maintenance requirement value, no provision for saltatory growth and new estimates of protein deposition identifies lower protein requirements than in previous reports. Higher values for adult amino acid requirements, derived from a re-evaluation of nitrogen balance studies and new stable isotope studies, identify some cereal-based diets as being inadequate for lysine. The main outstanding issues relate to the biological implausibility of the very low efficiencies of protein utilisation used in the factorial models for protein requirements for all population groups especially pregnancy when requirements may be overestimated. Also considerable uncertainty remains about the design and interpretation of most of the studies used to identify amino acid requirement values.

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

    Science.gov (United States)

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    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.

  5. 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.

  6. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

    Science.gov (United States)

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D.; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    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. PMID:25675104

  7. Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins.

    Science.gov (United States)

    Wang, Yang; Yi, Li; Wu, Zongfu; Shao, Jing; Liu, Guangjin; Fan, Hongjie; Zhang, Wei; Lu, Chengping

    2012-01-01

    Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.

  8. 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.

  9. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    Science.gov (United States)

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    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.

  10. 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.

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

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    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.

  12. Sequence Analysis of Hypothetical Proteins from Helicobacter pylori 26695 to Identify Potential Virulence Factors

    Science.gov (United States)

    Naqvi, Ahmad Abu Turab; Anjum, Farah; Khan, Faez Iqbal; Islam, Asimul; Ahmad, Faizan

    2016-01-01

    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. PMID:27729842

  13. Triadin/Junctin double null mouse reveals a differential role for Triadin and Junctin in anchoring CASQ to the jSR and regulating Ca(2+ homeostasis.

    Directory of Open Access Journals (Sweden)

    Simona Boncompagni

    Full Text Available Triadin (Tdn and Junctin (Jct are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ and ryanodine receptor (RyRs at the junctional sarcoplasmic reticulum (jSR. However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null, Jct (Jct-null or both (Tdn/Jct-null, we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+ imaging and Ca(2+ selective microelectrodes we found that changes in e-c coupling, SR Ca(2+content and resting [Ca(2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+ regulation than Jct/CASQ association.

  14. Identifying initial molecular targets of PDT: protein and lipid oxidation products

    Science.gov (United States)

    Oleinick, Nancy L.; Kim, Junhwan; Rodriguez, Myriam E.; Xue, Liang-yan; Kenney, Malcolm E.; Anderson, Vernon E.

    2009-06-01

    Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite. However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by PDT.

  15. Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

    Science.gov (United States)

    Mondal, Arindam; Victor, Ken G; Pudupakam, R S; Lyons, Charles E; Wertz, Gail W

    2014-02-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

  16. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    Science.gov (United States)

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  17. New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri

    Science.gov (United States)

    Alegria, Marcos C.; Docena, Cassia; Khater, Leticia; Ramos, Carlos H. I.; da Silva, Ana C. R.; Farah, Chuck S.

    2004-01-01

    We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity. PMID:15342589

  18. Nonspecific cytotoxic cell antimicrobial protein (NCAMP-1: a novel alarmin ligand identified in zebrafish.

    Directory of Open Access Journals (Sweden)

    Margaret Mariscal Monette

    Full Text Available Cells from the coelomic cavity of adult zebrafish (zf were used to study the alarmin-like activities of nonspecific cytotoxic cell antimicrobial protein-1 (NCAMP-1. Immunohistochemistry studies using polyclonal anti-NCAMP-1 identified constitutive NCAMP-1 in epithelial cells of the zf anterior kidney, in liver parenchyma and in the lamina propria of the intestine. NCAMP-1 was also located in the cytosol of mononuclear cells in these tissues. Cytosolic NCAMP-1 was detected in a diverse population of coelomic cells (CC using confocal microscopy and polyclonal anti-NCAMP-1 staining. Large mononuclear and heterophil-like CC had intracellular NCAMP-1. These studies indicated that NCAMP-1 is constitutively found in epithelial cells and in ZFCC. To establish a relationship between NCAMP-1 and the alarmin functions of ATP, a stimulation-secretion model was initiated using zf coelomic cells (ZFCC. ZFCCs treated with the alarmin ATP secreted NCAMP-1 into culture supernatants. Treatment of ZFCC with either ATP or NCAMP-1 activated purinergic receptor induced pore formation detected by the ZFCC uptake of the dye YO-PRO-1. ATP induced YO-PRO-1 uptake was inhibited by antagonists oxidized-ATP, KN62, or CBB. These antagonists did not compete with NCAMP-1 induced YO-PRO-1 uptake. Binding of ZFCC by both ATP and NCAMP-1 produced an influx of Ca2+. Combined treatment of ZFCC with ATP and NCAMP-1 increased target cell cytotoxicity. Individually NCAMP-1 or ATP treatment did not produce target cell damage. Similar to ATP, NCAMP-1 activates cellular pore formation, calcium influx and cytotoxicity.

  19. Nonspecific Cytotoxic Cell Antimicrobial Protein (NCAMP-1): A Novel Alarmin Ligand Identified in Zebrafish

    Science.gov (United States)

    Monette, Margaret Mariscal; Evans, Donald Lee; Krunkosky, Thomas; Camus, Alvin; Jaso-Friedmann, Liliana

    2015-01-01

    Cells from the coelomic cavity of adult zebrafish (zf) were used to study the alarmin-like activities of nonspecific cytotoxic cell antimicrobial protein-1 (NCAMP-1). Immunohistochemistry studies using polyclonal anti-NCAMP-1 identified constitutive NCAMP-1 in epithelial cells of the zf anterior kidney, in liver parenchyma and in the lamina propria of the intestine. NCAMP-1 was also located in the cytosol of mononuclear cells in these tissues. Cytosolic NCAMP-1 was detected in a diverse population of coelomic cells (CC) using confocal microscopy and polyclonal anti-NCAMP-1 staining. Large mononuclear and heterophil-like CC had intracellular NCAMP-1. These studies indicated that NCAMP-1 is constitutively found in epithelial cells and in ZFCC. To establish a relationship between NCAMP-1 and the alarmin functions of ATP, a stimulation-secretion model was initiated using zf coelomic cells (ZFCC). ZFCCs treated with the alarmin ATP secreted NCAMP-1 into culture supernatants. Treatment of ZFCC with either ATP or NCAMP-1 activated purinergic receptor induced pore formation detected by the ZFCC uptake of the dye YO-PRO-1. ATP induced YO-PRO-1 uptake was inhibited by antagonists oxidized-ATP, KN62, or CBB. These antagonists did not compete with NCAMP-1 induced YO-PRO-1 uptake. Binding of ZFCC by both ATP and NCAMP-1 produced an influx of Ca2+. Combined treatment of ZFCC with ATP and NCAMP-1 increased target cell cytotoxicity. Individually NCAMP-1 or ATP treatment did not produce target cell damage. Similar to ATP, NCAMP-1 activates cellular pore formation, calcium influx and cytotoxicity. PMID:25689842

  20. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

    Science.gov (United States)

    Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P; Henske, John K; O'Malley, Michelle A

    2016-12-20

    Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes

  1. Novel seminal fluid proteins in the seed beetle Callosobruchus maculatus identified by a proteomic and transcriptomic approach.

    Science.gov (United States)

    Bayram, H; Sayadi, A; Goenaga, J; Immonen, E; Arnqvist, G

    2017-02-01

    The seed beetle Callosobruchus maculatus is a significant agricultural pest and increasingly studied model of sexual conflict. Males possess genital spines that increase the transfer of seminal fluid proteins (SFPs) into the female body. As SFPs alter female behaviour and physiology, they are likely to modulate reproduction and sexual conflict in this species. Here, we identified SFPs using proteomics combined with a de novo transcriptome. A prior 2D-sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis identified male accessory gland protein spots that were probably transferred to the female at mating. Proteomic analysis of these spots identified 98 proteins, a majority of which were also present within ejaculates collected from females. Standard annotation workflows revealed common functional groups for SFPs, including proteases and metabolic proteins. Transcriptomic analysis found 84 transcripts differentially expressed between the sexes. Notably, genes encoding 15 proteins were highly expressed in male abdomens and only negligibly expressed within females. Most of these sequences corresponded to 'unknown' proteins (nine of 15) and may represent rapidly evolving SFPs novel to seed beetles. Our combined analyses highlight 44 proteins for which there is strong evidence that they are SFPs. These results can inform further investigation, to better understand the molecular mechanisms of sexual conflict in seed beetles.

  2. 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.

  3. Two-Dimensional Differential Gel Electrophoresis to Identify Protein Biomarkers in Amniotic Fluid of Edwards Syndrome (Trisomy 18 Pregnancies.

    Directory of Open Access Journals (Sweden)

    Te-Yao Hsu

    Full Text Available Edwards syndrome (ES is a severe chromosomal abnormality with a prevalence of about 0.8 in 10,000 infants born alive. The aims of this study were to identify candidate proteins associated with ES pregnancies from amniotic fluid supernatant (AFS using proteomics, and to explore the role of biological networks in the pathophysiology of ES.AFS from six second trimester pregnancies with ES fetuses and six normal cases were included in this study. Fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS were used for comparative proteomic analysis. The identified proteins were further validated by Western blotting and the role of biological networks was analyzed.Twelve protein spots were differentially expressed by more than 1.5-fold in the AFS of the ES pregnancies. MALDI-TOF/MS identified one up-regulated protein: apolipoprotein A1 (ApoA1, and four under-regulated proteins: vitamin D binding protein (VDBP, alpha-1-antitrypsin (A1AT, insulin-like growth factor-binding protein 1 (IGFBP-1, and transthyretin (TTR. Western blot and densitometric analysis of ApoA1, A1AT, IGFBP-1, and TTR confirmed the alteration of these proteins in the amniotic fluid samples. Biological network analysis revealed that the proteins of the ES AFS were involved mainly in lipid and hormone metabolism, immune response, and cardiovascular disease.These five proteins may be involved in the pathogenesis of ES. Further studies are needed to explore.

  4. Proteomics of photoreceptor outer segments identifies a subset of SNARE and Rab proteins implicated in membrane vesicle trafficking and fusion.

    Science.gov (United States)

    Kwok, Michael C M; Holopainen, Juha M; Molday, Laurie L; Foster, Leonard J; Molday, Robert S

    2008-06-01

    The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.

  5. 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.

  6. 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.

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

    NARCIS (Netherlands)

    van Dijk, David; Ertaylan, Gokhan; Boucher, Charles Ab; Sloot, Peter Ma

    2010-01-01

    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 dynamicall

  8. 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.

  9. Venom gland transcriptomics for identifying, cataloging, and characterizing venom proteins in snakes.

    Science.gov (United States)

    Brahma, Rajeev Kungur; McCleary, Ryan J R; Kini, R Manjunatha; Doley, Robin

    2015-01-01

    Snake venoms are cocktails of protein toxins that play important roles in capture and digestion of prey. Significant qualitative and quantitative variation in snake venom composition has been observed among and within species. Understanding these variations in protein components is instrumental in interpreting clinical symptoms during human envenomation and in searching for novel venom proteins with potential therapeutic applications. In the last decade, transcriptomic analyses of venom glands have helped in understanding the composition of various snake venoms in great detail. Here we review transcriptomic analysis as a powerful tool for understanding venom profile, variation and evolution.

  10. Mem-ADSVM: A two-layer multi-label predictor for identifying multi-functional types of membrane proteins.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2016-06-07

    Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. However, most of the existing membrane-protein predictors have the following problems: (1) they do not predict whether a given protein is a membrane protein or not; (2) they are limited to predicting membrane proteins with single-label functional types but ignore those with multi-functional types; and (3) there is still much room for improvement for their performance. To address these problems, this paper proposes a two-layer multi-label predictor, namely Mem-ADSVM, which can identify membrane proteins (Layer I) and their multi-functional types (Layer II). Specifically, given a query protein, its associated gene ontology (GO) information is retrieved by searching a compact GO-term database with its homologous accession number. Subsequently, the GO information is classified by a binary support vector machine (SVM) classifier to determine whether it is a membrane protein or not. If yes, it will be further classified by a multi-label multi-class SVM classifier equipped with an adaptive-decision (AD) scheme to determine to which functional type(s) it belongs. Experimental results show that Mem-ADSVM significantly outperforms state-of-the-art predictors in terms of identifying both membrane proteins and their multi-functional types. This paper also suggests that the two-layer prediction architecture is better than the one-layer for prediction performance. For reader׳s convenience, the Mem-ADSVM server is available online at http://bioinfo.eie.polyu.edu.hk/MemADSVMServer/.

  11. Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach in identifying a new functional split intein.

    Directory of Open Access Journals (Sweden)

    Yun-Tzai Lee

    Full Text Available Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein trans-splicing (PTS activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of in silico CP prediction facilitated the design of split intein as well as circular permutants.

  12. Self-Organizing Feature Maps Identify Proteins Critical to Learning in a Mouse Model of Down Syndrome

    Science.gov (United States)

    Higuera, Clara; Gardiner, Katheleen J.; Cios, Krzysztof J.

    2015-01-01

    Down syndrome (DS) is a chromosomal abnormality (trisomy of human chromosome 21) associated with intellectual disability and affecting approximately one in 1000 live births worldwide. The overexpression of genes encoded by the extra copy of a normal chromosome in DS is believed to be sufficient to perturb normal pathways and normal responses to stimulation, causing learning and memory deficits. In this work, we have designed a strategy based on the unsupervised clustering method, Self Organizing Maps (SOM), to identify biologically important differences in protein levels in mice exposed to context fear conditioning (CFC). We analyzed expression levels of 77 proteins obtained from normal genotype control mice and from their trisomic littermates (Ts65Dn) both with and without treatment with the drug memantine. Control mice learn successfully while the trisomic mice fail, unless they are first treated with the drug, which rescues their learning ability. The SOM approach identified reduced subsets of proteins predicted to make the most critical contributions to normal learning, to failed learning and rescued learning, and provides a visual representation of the data that allows the user to extract patterns that may underlie novel biological responses to the different kinds of learning and the response to memantine. Results suggest that the application of SOM to new experimental data sets of complex protein profiles can be used to identify common critical protein responses, which in turn may aid in identifying potentially more effective drug targets. PMID:26111164

  13. Anisotropic coarse-grained statistical potentials improve the ability to identify native-like protein structures

    CERN Document Server

    Buchete, N V; Thirumalai, D

    2003-01-01

    We present a new method to extract distance and orientation dependent potentials between amino acid side chains using a database of protein structures and the standard Boltzmann device. The importance of orientation dependent interactions is first established by computing orientational order parameters for proteins with alpha-helical and beta-sheet architecture. Extraction of the anisotropic interactions requires defining local reference frames for each amino acid that uniquely determine the coordinates of the neighboring residues. Using the local reference frames and histograms of the radial and angular correlation functions for a standard set of non-homologue protein structures, we construct the anisotropic pair potentials. The performance of the orientation dependent potentials was studied using a large database of decoy proteins. The results demonstrate that the new distance and orientation dependent residue-residue potentials present a significantly improved ability to recognize native folds from a set o...

  14. An in vivo crosslinking system for identifying mycobacterial protein–protein interactions

    Science.gov (United States)

    Lougheed, Kathryn E.A.; Bennett, Mark H.; Williams, Huw D.

    2014-01-01

    The analysis of protein–protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His–Strep–Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex. PMID:25034228

  15. 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

    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...

  16. 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.

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

    Science.gov (United States)

    Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    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. PMID:28006025

  18. SMpred: a support vector machine approach to identify structural motifs in protein structure without using evolutionary information.

    Science.gov (United States)

    Pugalenthi, Ganesan; Kandaswamy, Krishna Kumar; Suganthan, P N; Sowdhamini, R; Martinetz, Thomas; Kolatkar, Prasanna R

    2010-12-01

    Knowledge of three dimensional structure is essential to understand the function of a protein. Although the overall fold is made from the whole details of its sequence, a small group of residues, often called as structural motifs, play a crucial role in determining the protein fold and its stability. Identification of such structural motifs requires sufficient number of sequence and structural homologs to define conservation and evolutionary information. Unfortunately, there are many structures in the protein structure databases have no homologous structures or sequences. In this work, we report an SVM method, SMpred, to identify structural motifs from single protein structure without using sequence and structural homologs. SMpred method was trained and tested using 132 proteins domains containing 581 motifs. SMpred method achieved 78.79% accuracy with 79.06% sensitivity and 78.53% specificity. The performance of SMpred was evaluated with MegaMotifBase using 188 proteins containing 1161 motifs. Out of 1161 motifs, SMpred correctly identified 1503 structural motifs reported in MegaMotifBase. Further, we showed that SMpred is useful approach for the length deviant superfamilies and single member superfamilies. This result suggests the usefulness of our approach for facilitating the identification of structural motifs in protein structure in the absence of sequence and structural homologs. The dataset and executable for the SMpred algorithm is available at http://www3.ntu.edu.sg/home/EPNSugan/index_files/SMpred.htm.

  19. The gelsolin/fragmin family protein identified in the higher plant Mimosa pudica.

    Science.gov (United States)

    Yamashiro, S; Kameyama, K; Kanzawa, N; Tamiya, T; Mabuchi, I; Tsuchiya, T

    2001-08-01

    Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)-dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca(2+)-dependent manner.

  20. A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data.

    Science.gov (United States)

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2016-02-06

    The Golgi Apparatus (GA) is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP), a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE) is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE) is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF) module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew's Correlation Coefficient (MCC) of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions.

  1. A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2016-02-01

    Full Text Available The Golgi Apparatus (GA is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP, a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew’s Correlation Coefficient (MCC of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions.

  2. Topological analysis of protein co-abundance networks identifies novel host targets important for HCV infection and pathogenesis

    Directory of Open Access Journals (Sweden)

    McDermott Jason E

    2012-04-01

    Full Text Available Abstract Background High-throughput methods for obtaining global measurements of transcript and protein levels in biological samples has provided a large amount of data for identification of 'target' genes and proteins of interest. These targets may be mediators of functional processes involved in disease and therefore represent key points of control for viruses and bacterial pathogens. Genes and proteins that are the most highly differentially regulated are generally considered to be the most important. We present topological analysis of co-abundance networks as an alternative to differential regulation for confident identification of target proteins from two related global proteomics studies of hepatitis C virus (HCV infection. Results We analyzed global proteomics data sets from a cell culture study of HCV infection and from a clinical study of liver biopsies from HCV-positive patients. Using lists of proteins known to be interaction partners with pathogen proteins we show that the most differentially regulated proteins in both data sets are indeed enriched in pathogen interactors. We then use these data sets to generate co-abundance networks that link proteins based on similar abundance patterns in time or across patients. Analysis of these co-abundance networks using a variety of network topology measures revealed that both degree and betweenness could be used to identify pathogen interactors with better accuracy than differential regulation alone, though betweenness provides the best discrimination. We found that though overall differential regulation was not correlated between the cell culture and liver biopsy data, network topology was conserved to an extent. Finally, we identified a set of proteins that has high betweenness topology in both networks including a protein that we have recently shown to be essential for HCV replication in cell culture. Conclusions The results presented show that the network topology of protein co

  3. A method for identifying a proposed carbohydrate-binding motif of proteins.

    Science.gov (United States)

    Baumann, M A; Anderson, B E

    1991-11-01

    An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E. coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate. These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure. An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria. The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence. When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

    DEFF Research Database (Denmark)

    Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P.;

    2016-01-01

    machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display...... a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce....

  5. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    Science.gov (United States)

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

  6. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    Science.gov (United States)

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  7. Large-scale proteomics differentiates cholesteatoma from surrounding tissues and identifies novel proteins related to the pathogenesis.

    Directory of Open Access Journals (Sweden)

    Anders Britze

    Full Text Available Cholesteatoma is the growth of keratinizing squamous epithelium in the middle ear. It is associated with severe complications and has a poorly understood etiopathogenesis. Here, we present the results from extensive bioinformatics analyses of the first large-scale proteomic investigation of cholesteatoma. The purpose of this study was to take an unbiased approach to identifying alterations in protein expression and in biological processes, in order to explain the characteristic phenotype of this skin-derived tumor. Five different human tissue types (cholesteatoma, neck of cholesteatoma, tympanic membrane, external auditory canal skin, and middle ear mucosa were analyzed. More than 2,400 unique proteins were identified using nanoLC-MS/MS based proteomics (data deposited to the ProteomeXchange, and 295 proteins were found to be differentially regulated in cholesteatoma. Validation analyses were performed by SRM mass spectrometry. Proteins found to be up- or down-regulated in cholesteatoma were analyzed using Ingenuity Pathway Analysis and clustered into functional groups, for which activation state and associations to disease processes were predicted. Cholesteatoma contained high levels of pro-inflammatory S100 proteins, such as S100A7A and S100A7. Several proteases, such as ELANE, were up-regulated, whereas extracellular matrix proteins, such as COL18A1 and NID2, were under-represented. This may lead to alterations in integrity and differentiation of the tissue (as suggested by the up-regulation of KRT4 in the cholesteatoma. The presented data on the differential protein composition in cholesteatoma corroborate previous studies, highlight novel protein functionalities involved in the pathogenesis, and identify new areas for targeted research that hold therapeutic potential for the disease.

  8. 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

  9. Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination.

    Science.gov (United States)

    Kegel, Linde; Jaegle, Martine; Driegen, Siska; Aunin, Eerik; Leslie, Kris; Fukata, Yuko; Watanabe, Masahiko; Fukata, Masaki; Meijer, Dies

    2014-04-01

    The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.

  10. Transient changes in intercellular protein variability identify sources of noise in gene expression.

    Science.gov (United States)

    Singh, Abhyudai

    2014-11-01

    Protein levels differ considerably between otherwise identical cells, and these differences significantly affect biological function and phenotype. Previous work implicated various noise mechanisms that drive variability in protein copy numbers across an isogenic cell population. For example, transcriptional bursting of mRNAs has been shown to be a major source of noise in the expression of many genes. Additional expression variability, referred to as extrinsic noise, arises from intercellular variations in mRNA transcription and protein translation rates attributed to cell-to-cell differences in cell size, abundance of ribosomes, etc. We propose a method to determine the magnitude of different noise sources in a given gene of interest. The method relies on blocking transcription and measuring changes in protein copy number variability over time. Our results show that this signal has sufficient information to quantify both the extent of extrinsic noise and transcription bursting in gene expression. Moreover, if the mean mRNA count is known, then the relative contributions of transcription versus translation rate fluctuations to extrinsic noise can also be determined. In summary, our study provides an easy-to-implement method for characterizing noisy protein expression that complements existing techniques for studying stochastic dynamics of genetic circuits.

  11. Clinical features and functional significance of the P369S/R408Q variant in pyrin, the familial Mediterranean fever protein

    Science.gov (United States)

    Ryan, JG; Masters, SL; Booty, MG; Habal, N; Alexander, JD; Barham, BK; Remmers, EF; Barron, KS; Kastner, DL; Aksentijevich, I

    2013-01-01

    Objectives Familial Mediterranean fever (FMF) is caused by mutations in MEFV, which encodes pyrin. The nature of substitutions P369S and R408Q in exon 3 remains unclear. Exon 3 encoding pyrin’s B-box domain is necessary for interactions with PSTPIP1. We aimed to characterize the phenotype of patients with these substitutions and to determine their functional significance. Methods A database of genetic tests undertaken in our institution was interrogated. Symptoms and signs were classified according to Tel-Hashomer criteria. Co-immunoprecipation techniques were employed to determine the variants’ effects on pyrin/PSTPIP1 interactions. Results We identified 40 symptomatic and 4 asymptomatic family members with these substitutions. P369S and R408Q were found in cis, and co-segregated in all patients sequenced. Clinical details were available on 22 patients. Five patients had symptoms and signs fulfilling a clinical diagnosis of FMF. Fourteen received colchicine. In patients not reaching the criteria, trials of anti-TNF agents resulted in partial or no benefit; resolution of symptoms was noted in those receiving anakinra. The carrier frequency was higher in the patient cohort than in controls but was not statistically significant. Co-immunoprecipitation studies demonstrated that these pyrin variants did not affect binding to PSTPIP1. Conclusions P369S/R408Q substitutions are associated with a highly variable phenotype, and are infrequently associated with typical FMF symptoms, however a trial of colchicine is warranted in all. Functional and modeling studies suggest that these substitutions do not significantly affect pyrin’s interaction with PSTPIP1. This study highlights the need for caution in interpreting genetic tests in patients with atypical symptoms. PMID:19934105

  12. Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

    Science.gov (United States)

    Lu, Jing; Boeren, S; de Vries, S C; van Valenberg, H J F; Vervoort, J; Hettinga, K

    2011-12-10

    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 environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Charges in Hydrophobic Environments: A Strategy for Identifying Alternative States in Proteins.

    Science.gov (United States)

    Robinson, Aaron C; Majumdar, Ananya; Schlessman, Jamie L; García-Moreno E, Bertrand

    2017-01-10

    In the V23E variant of staphylococcal nuclease, Glu-23 has a pKa of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands β1 and β2 separate from the β-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent pKa of an internal ionizable group can reflect the average of the very different pKa values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the pKa value of internal ionizable group reflects conformational reorganization. (3) The apparent pKa of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict pKa values of internal residues and to reproduce pH-driven conformational transitions of proteins.

  14. 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.

  15. A tyrosine-rich cell surface protein in the diatom Amphora coffeaeformis identified through transcriptome analysis and genetic transformation.

    Directory of Open Access Journals (Sweden)

    Matthias T Buhmann

    Full Text Available Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis.

  16. 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...

  17. Identifying Ca2+-Binding Sites in Proteins by Liquid Chromatography-Mass Spectrometry Using Ca2+-Directed Dissociations

    NARCIS (Netherlands)

    Jamalian, Azadeh; Sneekes, Evert-Jan; Wienk, Hans; Dekker, Lennard J. M.; Ruttink, Paul J. A.; Ursem, Mario; Luider, Theo M.; Burgers, Peter C.

    2014-01-01

    Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry app

  18. 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...... spectrometer operated in data-dependent acquisition mode. RESULTS: We identified a total of 35,664 unique peptides from 4,161 different proteins, of which 1,946 and 2,090 were of bacterial and human origin, respectively. The human protein profiles displayed significant overexpression of the complement system...

  19. New classes of mind bomb-interacting proteins identified from yeast two-hybrid screens.

    Science.gov (United States)

    Tseng, Li-Chuan; Zhang, Chengjin; Cheng, Chun-Mei; Xu, Haoying; Hsu, Chia-Hao; Jiang, Yun-Jin

    2014-01-01

    Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.

  20. 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.

  1. [Protein expression patterns identify morphological variability of ascomatal hairs in two species of genus Chaetomium].

    Science.gov (United States)

    Sun, Qin; Liu, Xiaoyong; Wang, Qian; Luo, Yuanming; Wang, Xuewei

    2012-10-04

    Morphology of ascomatal hairs was traditionally used as a primary character in the classification of the fungal genus Chaetomium. However, the taxonomic value of ascomatal hair morphology is questioned in modern taxonomy of Chaetomium. Chaetomium indicum and C. funicola are two species proposed only by their differences in ascomatal hairs. The aim of this study is to understand the difference between these two species and their variability in the morphology of ascomatal hairs at the level of protein expression patterns, as well as to ressess the taxonomic value of the ascomatal hairs. We performed microscopic examination to obtain the morphological characters of the typical and variable strains in both C. indicum and C. funicola. Then we used two-dimensional gel electrophoresis (2DE) to compare the protein expression patterns of the two species, including their typical and variable strains. The comparison of the obtained 2DE maps indicated that C. indicum and C. funicola exhibited species-specific protein expression patterns. The phylogenetic tree derived from the distance matrix of expression patterns with Neighbor-joining algorithm also revealed that the tested strains of C. indicum and C. funicola fell into two distinct clades, among which the variant strains were grouped together with the typical strains of the same species. The consistency of species delimitation between C. indicum and C. funicola based on morphological characters of ascomatal hairs and species-specific protein expression patterns demonstrates that ascomatal hairs can be still used as potential morphological parameters in taxonomy of Chaetomium.

  2. 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, Toon A B; 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 hav

  3. Effect of newly identified hTERT-interacting proteins on telomerase activity

    Institute of Scientific and Technical Information of China (English)

    Lina Zhou; Bing Chen; Xing Hua; Ping Zhou; Lian Guo; Yong Peng; Kunhua Qiu

    2013-01-01

    There is a close relationship between telomeres-telomerase and age-related disease.Human telomerase reverse transcriptase (hTERT) is both the catalytic component of human telomerase and the rate-limiting determinant of telomerase activity.Its transcriptional regulation is the primary mode of control of telomerase activity.It is critical to find the proteins interacting with hTERT for exploring the regulatory mechanisms of the hTERT expression and the telomerase activity.In this study,the yeast two-hybrid system was used to screen the potential interactive proteins of hTERT.Six proteins were obtained,among which TSTAR,LOXL3,HKR3,and Par-4 were further confirmed as the interacting proteins of hTERT by co-immunopreci-pitation.Then the sense and antisense gene eukaryotic expression vectors containing these four genes were constructed and transfected into tumor cell lines.The correlations among the expression levels of these four proteins,the expression level of hTERT,and the telomerase activity were analyzed.Results showed that the up-regulation of TSTAR expression and down-regulation of HKR3 expression led to the increase of hTERT expression and telomerase activity,while the up-and down-regulation of LOXL3 and Par-4 expressions had no obvious effect.Our results suggested that T-STAR has a positive correlation with the telomerase activity while HKR3 may be a negative regulator.This conclusion is important to further explore the influencing factors or regulation pathways of human telomerase activity,which may be of great importance for the potential clinical application.

  4. RIBFIND: a web server for identifying rigid bodies in protein structures and to aid flexible fitting into cryo EM maps.

    Science.gov (United States)

    Pandurangan, Arun Prasad; Topf, Maya

    2012-09-15

    To better analyze low-resolution cryo electron microscopy maps of macromolecular assemblies, component atomic structures frequently have to be flexibly fitted into them. Reaching an optimal fit and preventing the fitting process from getting trapped in local minima can be significantly improved by identifying appropriate rigid bodies (RBs) in the fitted component. Here we present the RIBFIND server, a tool for identifying RBs in protein structures. The server identifies RBs in proteins by calculating spatial proximity between their secondary structural elements. The RIBFIND web server and its standalone program are available at http://ribfind.ismb.lon.ac.uk. a.pandurangan@mail.cryst.bbk.ac.uk Supplementary data are available at Bioinformatics online.

  5. 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.

  6. Proteomic analysis identifies proteins related to carotenoid accumulation in Yesso scallop (Patinopecten yessoensis).

    Science.gov (United States)

    Zhang, Yueyue; Zhang, Lingling; Sun, Jin; Qiu, Jianwen; Hu, Xiaoli; Hu, Jingjie; Bao, Zhenmin

    2014-03-15

    Carotenoids are powerful antioxidants that affect many physiological functions. As an important source of natural carotenoids, marine mollusks contain various types of carotenoids and are receiving increasing research attention. To better understand the molecular mechanism underlying carotenoid accumulation in marine mollusks, a new variety of carotenoid-enriched Yesso scallop (Patinopecten yessoensis), named "Haida golden scallop", was used in this study. A proteomic approach was applied to explore the differences between the new variety and common individuals, resulting in seven differentially expressed proteins. Real-time PCR showed that four of the corresponding genes were also significantly up-regulated at the mRNA level in the new variety. Genes involved in various biological processes, such as lipid and glucose metabolism, protein-folding and degradation, were altered. Peroxisome proliferator-activated receptors (PPARs) may play a vital role in these changes. This study represents the first step towards future work on the genetic basis of carotenoid accumulation in marine mollusks.

  7. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    OpenAIRE

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nucl...

  8. 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.

  9. Empirical Methods for Identifying Specific Peptide-protein Interactions for Smart Reagent Development

    Science.gov (United States)

    2012-09-01

    library as C-terminal fusion to the capsid protein D of bacteriophage lambda,” J Mol Biol, 282(1), 125-35 (1998). [10] Z. Ren, and L. W. Black, “Phage T4...Blockmans et al., “Clinical and serological evaluation of a novel CENP-A peptide based ELISA,” Arthritis Research & Therapy , 12(3), R99-R113 (2010). [13

  10. The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.

    Science.gov (United States)

    Perreault, Andrea A; Venters, Bryan J

    2016-12-23

    Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

  11. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    Science.gov (United States)

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin.

  12. Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs.

    Science.gov (United States)

    Park, Jong-Hwa; Back, Jung Ho; Hahm, Soo Hyun; Shim, Hye-Young; Park, Min Ju; Ko, Sung Il; Han, Ye Sun

    2007-10-01

    We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

  13. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis.

    Science.gov (United States)

    Culver, Brady P; Savas, Jeffrey N; Park, Sung K; Choi, Jeong H; Zheng, Shuqiu; Zeitlin, Scott O; Yates, John R; Tanese, Naoko

    2012-06-22

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.

  14. Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

    Science.gov (United States)

    den Reijer, P Martijn; Sandker, Marjan; Snijders, Susan V; Tavakol, Mehri; Hendrickx, Antoni P A; van Wamel, Willem J B

    2017-02-01

    Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

  15. QuatIdent: a web server for identifying protein quaternary structural attribute by fusing functional domain and sequential evolution information.

    Science.gov (United States)

    Shen, Hong-Bin; Chou, Kuo-Chen

    2009-03-01

    Many proteins exist in vivo as oligomers with various different quaternary structural attributes rather than as single individual chains. They are the structural bases of various marvelous biological functions such as cooperative effects, allosteric mechanism, and ion-channel gating. Therefore, with the avalanche of protein sequences generated in the postgenomic era, it is very important for both basic research and drug discovery to identify their quaternary structural attributes in a timely manner. In view of this, a powerful ensemble identifier, called QuatIdent, is developed by fusing the functional domain and sequential evolution information. QuatIdent is a 2-layer predictor. The 1st layer is for identifying a query protein as belonging to which one of the following 10 main quaternary structural attributes: (1) monomer, (2) dimer, (3) trimer, (4) tetramer, (5) pentamer, (6) hexamer, (7) heptamer, (8) octamer, (9) decamer, and (10) dodecamer. If the result thus obtained turns out to be anything but monomer, the process will be automatically continued to further identify it as belonging to a homo-oligomer or hetero-oligomer. The overall success rate by QuatIdent for the 1st layer identification was 71.1% and that for the 2nd layer ranged from 84 to 96%. These rates were derived by the jackknife cross-validation tests on the stringent benchmark data sets where none of proteins has > or =60% pairwise sequence identity to any other in a same subset. QuatIdent is freely accessible to the public as a web server via the site at http://www.csbio.sjtu.edu.cn/bioinf/Quaternary/ , by which one can get the desired 2-level results for a query protein sequence in around 25 seconds. The longer the sequence is, the more time that is needed.

  16. 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.

  17. Mapping of Chikungunya virus interactions with host proteins identified nsP2 as a highly connected viral component.

    Science.gov (United States)

    Bouraï, Mehdi; Lucas-Hourani, Marianne; Gad, Hans Henrik; Drosten, Christian; Jacob, Yves; Tafforeau, Lionel; Cassonnet, Patricia; Jones, Louis M; Judith, Delphine; Couderc, Thérèse; Lecuit, Marc; André, Patrice; Kümmerer, Beate Mareike; Lotteau, Vincent; Desprès, Philippe; Tangy, Frédéric; Vidalain, Pierre-Olivier

    2012-03-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.

  18. 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.

  19. 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...

  20. Comprehensive Antigen Screening Identifies Moraxella catarrhalis Proteins That Induce Protection in a Mouse Pulmonary Clearance Model

    NARCIS (Netherlands)

    M. Smidt (Margarita); P. Bättig (Patrick); S.J.C. Verhaegh (Suzanne); A. Niebisch (Axel); M. Hanner (Markus); S. Selak (Sanja); W. Schüler (Wolfgang); E. Morfeldt (Eva); C. Hellberg (Christel); E. Nagy (Eszter); U. Lundberg (Urban); J.P. Hays (John); A. Meinke (Andreas); B. Henriques-Normark (Birgitta)

    2013-01-01

    textabstractMoraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with ot

  1. Transcriptional profiling of the two-component regulatory system VraSR in Staphylococcus aureus with low-level vancomycin resistance.

    Science.gov (United States)

    Chen, Hongbin; Xiong, Zhujia; Liu, Kuoyue; Li, Shuguang; Wang, Ruobing; Wang, Xiaojuan; Zhang, Yawei; Wang, Hui

    2016-05-01

    The objective of this study was to comprehensively identify the target genes regulated by the two-component regulatory system VraSR in Staphylococcus aureus and to clarify the role of VraSR in low-level vancomycin resistance. Expression of vraS was determined by real-time quantitative reverse transcriptase PCR (qRT-PCR). A clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain B6D and a vancomycin-intermediate S. aureus (VISA) strain D7 that was induced from a meticillin-resistant S. aureus strain were selected to construct vraSR null mutants by allelic replacement. The vraSR-complemented strain B6D_c was also constructed by allelic replacement. Genes differentially expressed in the wild-type, vraSR null mutant and complemented strains were detected using RNA-Seq and were validated by qRT-PCR. Compared with vancomycin-susceptible S. aureus strains, expression of vraS was upregulated in all four isogenic hVISA strains. Vancomycin minimum inhibitory concentrations (MICs) in the vraSR null mutants B6D-ΔvraSR and D7-ΔvraSR were significantly lower than in the wild-type strains B6D and D7 and the complemented strain B6D_c. RNA-Seq and qRT-PCR data showed that expression of genes encoding FmtA protein, foldase protein PrsA, capsular polysaccharide biosynthesis glycosyltransferase, TcaA, a putative membrane protein, and six hypothetical proteins was down regulated in both vraSR-null mutants B6D-ΔvraSR and D7-ΔvraSR. Most of these differentially expressed proteins are involved in cell wall biosynthesis, which is associated with vancomycin resistance in S. aureus. In conclusion, VraSR plays an important role in S. aureus strains with low-level vancomycin resistance. PrsA, FmtA, glycosyltransferase and TcaA are regulated directly or indirectly by VraSR.

  2. 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

  3. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    Science.gov (United States)

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

  4. 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.

  5. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry

    Directory of Open Access Journals (Sweden)

    Gisa Gerold

    2015-08-01

    Full Text Available Hepatitis C virus (HCV enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1, which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

  6. ICP35 Is a TREX-Like Protein Identified in White Spot Syndrome Virus

    Science.gov (United States)

    Phairoh, Panapat; Suthibatpong, Thana; Rattanarojpong, Triwit; Jongruja, Nujarin; Senapin, Saengchan; Choowongkomon, Kiattawee; Khunrae, Pongsak

    2016-01-01

    ICP35 is a non-structural protein from White spot syndrome virus believed to be important in viral replication. Since ICP35 was found to localize in the host nucleus, it has been speculated that the function of ICP35 might be involved in the interaction of DNA. In this study, we overexpressed, purified and characterized ICP35. The thioredoxin-fused ICP35 (thio-ICP35) was strongly expressed in E. coli and be able to form itself into dimers. Investigation of the interaction between ICP35 and DNA revealed that ICP35 can perform DNase activity. Structural model of ICP35 was successfully built on TREX1, suggesting that ICP35 might adopt the folding similar to that of TREX1 protein. Several residues important for dimerization in TREX1 are also conserved in ICP35. Residue Asn126 and Asp132, which are seen to be in close proximity to metal ions in the ICP35 model, were shown through site-directed mutagenesis to be critical for DNase activity. PMID:27348862

  7. Identifying constituents of whey protein concentrates that reduce the pink color defect in cooked ground turkey.

    Science.gov (United States)

    Sammel, L M; Claus, J R; Greaser, M L; Lucey, J A

    2007-12-01

    Whey protein concentrate constituents were tested for their ability to reduce naturally occurring pink color defect and pink cooked color induced by sodium nitrite (10ppm) and nicotinamide (1.0%) in ground turkey. β-lactoglobulin (1.8%), α-lactalbumin (0.8%), bovine serum albumin (0.15-0.3%), lactose (1.0-3.0%), potassium chloride (500-1500ppm), and ferrous iron chloride (0.3-30ppm) had no effects on cooked pink color. Lactoferrin (30-5000ppm) increased or decreased pink color depending on its concentration in samples without added sodium nitrite or nicotinamide. Annatto (0.1-1.0ppm) reduced pink color whereas the higher concentration of magnesium chloride (22-88ppm) and ferric iron chloride (0.3-30ppm) increased pink color in samples with added nicotinamide. Calcium chloride (160-480ppm) was the only tested constituent that consistently reduced pink cooked color in samples with and without added nitrite and nicotinamide. Due to the variability of whey protein concentrates and the number of constituents that do not reduce pink cooked color, the addition of calcium alone or dried milk minerals containing calcium, phosphate, and citrate, represents a better means to regularly prevent the pink color defect in cooked ground turkey.

  8. Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product.

    Science.gov (United States)

    Brundage, Elizabeth A; Biesiadecki, Brandon J; Reiser, Peter J

    2015-10-01

    Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, "masticatory", isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (a jaw-closing muscle), tibialis (predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (γTpm) isoforms (nomenclature reflects predominant tissue expression ("St"-striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (δTpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3.12 isoforms. We

  9. Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen.

    Science.gov (United States)

    Hill, Ryan C; Wither, Matthew J; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C

    2015-07-01

    Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼ 120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.

  10. Identifying differentially expressed proteins in two-dimensional electrophoresis experiments: inputs from transcriptomics statistical tools.

    Science.gov (United States)

    Artigaud, Sébastien; Gauthier, Olivier; Pichereau, Vianney

    2013-11-01

    Two-dimensional electrophoresis is a crucial method in proteomics that allows the characterization of proteins' function and expression. This usually implies the identification of proteins that are differentially expressed between two contrasting conditions, for example, healthy versus diseased in human proteomics biomarker discovery and stressful conditions versus control in animal experimentation. The statistical procedures that lead to such identifications are critical steps in the 2-DE analysis workflow. They include a normalization step and a test and probability correction for multiple testing. Statistical issues caused by the high dimensionality of the data and large-scale multiple testing have been a more active topic in transcriptomics than proteomics, especially in microarray analysis. We thus propose to adapt innovative statistical tools developed for microarray analysis and incorporate them in the 2-DE analysis pipeline. In this article, we evaluate the performance of different normalization procedures, different statistical tests and false discovery rate calculation methods with both real and simulated datasets. We demonstrate that the use of statistical procedures adapted from microarrays lead to notable increase in power as well as a minimization of false-positive discovery rate. More specifically, we obtained the best results in terms of reliability and sensibility when using the 'moderate t-test' from Smyth in association with classic false discovery rate from Benjamini and Hochberg. The methods discussed are freely available in the 'prot2D' open source R-package from Bioconductor (http://www.bioconductor.org//) under the terms of the GNU General Public License (version 2 or later). sebastien.artigaud@univ-brest.fr or sebastien.artigaud@gmx.com.

  11. Latent TGFβ binding protein 3 identifies a second heart field in zebrafish

    Science.gov (United States)

    Zhou, Yong; Cashman, Timothy J.; Nevis, Kathleen R.; Obregon, Pablo; Carney, Sara A.; Liu, Yan; Gu, Aihua; Mosimann, Christian; Sondalle, Samuel; Peterson, Richard E.; Heideman, Warren; Burns, Caroline E.; Burns, C. Geoffrey

    2012-01-01

    The four-chambered mammalian heart develops from two fields of cardiac progenitor cells (CPCs) distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart [1–3]. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract (OFT) and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary OFT but not a right ventricle [4], the existence and function of SHF-like cells in these species has remained a topic of speculation [4–10]. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss of function studies in zebrafish, a lower vertebrate with a single ventricle, that latent-TGFβ binding protein 3 (ltbp3) transcripts mark a field of CPCs with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the OFT and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGFβ ligands [11], zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of CPCs in both fields [4]. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Additionally, small-molecule inhibition of TGFβ signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGFβ type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGFβ signaling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species. PMID:21623370

  12. Combining ancestral sequence reconstruction with protein design to identify an interface hotspot in a key metabolic enzyme complex.

    Science.gov (United States)

    Holinski, Alexandra; Heyn, Kristina; Merkl, Rainer; Sterner, Reinhard

    2017-02-01

    It is important to identify hotspot residues that determine protein-protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA-HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA-HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site-directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high-affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. Proteins 2017; 85:312-321. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Quantitative Proteomic Analyses Identify ABA-Related Proteins and Signal Pathways in Maize Leaves under Drought Conditions.

    Science.gov (United States)

    Zhao, Yulong; Wang, Yankai; Yang, Hao; Wang, Wei; Wu, Jianyu; Hu, Xiuli

    2016-01-01

    Drought stress is one of major factors resulting in maize yield loss. The roles of abscisic acid (ABA) have been widely studied in crops in response to drought stress. However, more attention is needed to identify key ABA-related proteins and also gain deeper molecular insights about drought stress in maize. Based on this need, the physiology and proteomics of the ABA-deficient maize mutant vp5 and its wild-type Vp5 under drought stress were examined and analyzed. Malondialdehyde content increased and quantum efficiency of photosystem II decreased under drought stress in both genotypes. However, the magnitude of the increase or decrease was significantly higher in vp5 than in Vp5. A total of 7051 proteins with overlapping expression patterns among three replicates in the two genotypes were identified by Multiplex run iTRAQ-based quantitative proteomic and liquid chromatography-tandem mass spectrometry methods, of which the expression of only 150 proteins (130 in Vp5, 27 in vp5) showed changes of at least 1.5-fold under drought stress. Among the 150 proteins, 67 and 60 proteins were up-regulated and down-regulated by drought stress in an ABA-dependent way, respectively. ABA was found to play active roles in regulating signaling pathways related to photosynthesis, oxidative phosphorylation (mainly related to ATP synthesis), and glutathione metabolism (involved in antioxidative reaction) in the maize response to drought stress. Our results provide an extensive dataset of ABA-dependent, drought-regulated proteins in maize plants, which may help to elucidate the underlying mechanisms of ABA-enhanced tolerance to drought stress in maize.

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

    Science.gov (United States)

    Omer, Hélène; Alpha-Bazin, Béatrice; Brunet, Jean-Luc; Armengaud, Jean; Duport, Catherine

    2015-01-01

    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.

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

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    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.

  16. Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein

    Science.gov (United States)

    Liu, Chien-Hung; Zhou, Ligang; Chen, Guifang; Krug, Robert M.

    2015-01-01

    Previous studies showed that ZAPL (PARP-13.1) exerts its antiviral activity via its N-terminal zinc fingers that bind the mRNAs of some viruses, leading to mRNA degradation. Here we identify a different antiviral activity of ZAPL that is directed against influenza A virus. This ZAPL antiviral activity involves its C-terminal PARP domain, which binds the viral PB2 and PA polymerase proteins, leading to their proteasomal degradation. After the PB2 and PA proteins are poly(ADP-ribosylated), they are associated with the region of ZAPL that includes both the PARP domain and the adjacent WWE domain that is known to bind poly(ADP-ribose) chains. These ZAPL-associated PB2 and PA proteins are then ubiquitinated, followed by proteasomal degradation. This antiviral activity is counteracted by the viral PB1 polymerase protein, which binds close to the PARP domain and causes PB2 and PA to dissociate from ZAPL and escape degradation, explaining why ZAPL only moderately inhibits influenza A virus replication. Hence influenza A virus has partially won the battle against this newly identified ZAPL antiviral activity. Eliminating PB1 binding to ZAPL would be expected to substantially increase the inhibition of influenza A virus replication, so that the PB1 interface with ZAPL is a potential target for antiviral development. PMID:26504237

  17. Assessing the role of submarine groundwater discharge as a source of Sr to the Mediterranean Sea

    Science.gov (United States)

    Trezzi, Giada; Garcia-Orellana, Jordi; Rodellas, Valentí; Masqué, Pere; Garcia-Solsona, Ester; Andersson, Per S.

    2017-03-01

    Submarine groundwater discharge (SGD) has been identified as an important source of Sr to the ocean and the SGD-driven Sr flux to the global ocean has been recently re-evaluated (Beck et al., 2013). However, the uncertainty of this value is still high because of the uncertainties related to the determination of SGD flow rates and the paucity of 87Sr/86Sr data in SGD end-members. As carbonates have high Sr concentrations and are subjected to intense heightened weathering, they might significantly influence the SGD input of Sr to the ocean. Here we present data on Sr concentrations and 87Sr/86Sr ratios in three carbonate dominated sites of the western area of the Mediterranean Sea, a semi-enclosed basin characterized by abundant coastal carbonates. The 87Sr/86Sr ratios in groundwater were lower compared to modern seawater (∼0.70916), as expected for areas dominated by carbonate lithologies. Concentrations of Sr and 87Sr/86Sr ratios in groundwater showed conservative mixing in the studied subterranean estuaries. By using SGD flow rates reported in the literature for the study areas, a flow-weighted fresh SGD end-member characterized by a Sr concentration of 27-30 μM and a 87Sr/86Sr ratio of 0.707834-0.708020 was calculated for the eastern coast of the Iberian Peninsula. Integrating these Sr data with literature data (i.e. values of Sr concentration and 87Sr/86Sr ratio from other lithologies as well as SGD flow rates), we also calculated the fresh SGD-driven Sr flux to the entire Mediterranean Sea, obtaining a value of (0.34-0.83)·109 mol y-1, with a 87Sr/86Sr of 0.7081-0.7086. Thus, for the entire Mediterranean basin, SGD is globally a source of Sr less radiogenic compared to seawater. The SGD Sr flux to the Mediterranean Sea represents 5-6% of the SGD Sr flux to the global ocean and the Mediterranean SGD end-member has higher Sr concentration (5-12 μM) than the global SGD end-member (2.9 μM). This confirms the significant role of carbonate lithologies on SGD

  18. Using the yeast three-hybrid system to identify proteins that interact with a phloem-mobile mRNA

    Directory of Open Access Journals (Sweden)

    Sung Ki eCho

    2012-08-01

    Full Text Available Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3´ untranslated region (UTR of the RNA in mediating transport. Because the 3´ UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3´ UTR of StBEL5 as bait in the yeast three-hybrid system to identify putative partner RNA-binding proteins (RBPs. From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3´ UTR of StBEL5 using β-galactosidase assays in the yeast three-hybrid system and RNA gel-shift assays. Among the final selections were two RNA-binding proteins, a zinc-finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the yeast three-hybrid system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RNA-binding proteins. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

  19. Capture compound mass spectrometry--a powerful tool to identify novel c-di-GMP effector proteins.

    Science.gov (United States)

    Laventie, Benoît-Joseph; Nesper, Jutta; Ahrné, Erik; Glatter, Timo; Schmidt, Alexander; Jenal, Urs

    2015-01-01

    Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail. We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms(1). Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

  20. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    Directory of Open Access Journals (Sweden)

    Sara Landeras-Bueno

    2016-04-01

    Full Text Available Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.

  1. 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 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.

  2. iPPBS-Opt: A Sequence-Based Ensemble Classifier for Identifying Protein-Protein Binding Sites by Optimizing Imbalanced Training Datasets

    Directory of Open Access Journals (Sweden)

    Jianhua Jia

    2016-01-01

    Full Text Available Knowledge of protein-protein interactions and their binding sites is indispensable for in-depth understanding of the networks in living cells. With the avalanche of protein sequences generated in the postgenomic age, it is critical to develop computational methods for identifying in a timely fashion the protein-protein binding sites (PPBSs based on the sequence information alone because the information obtained by this way can be used for both biomedical research and drug development. To address such a challenge, we have proposed a new predictor, called iPPBS-Opt, in which we have used: (1 the K-Nearest Neighbors Cleaning (KNNC and Inserting Hypothetical Training Samples (IHTS treatments to optimize the training dataset; (2 the ensemble voting approach to select the most relevant features; and (3 the stationary wavelet transform to formulate the statistical samples. Cross-validation tests by targeting the experiment-confirmed results have demonstrated that the new predictor is very promising, implying that the aforementioned practices are indeed very effective. Particularly, the approach of using the wavelets to express protein/peptide sequences might be the key in grasping the problem’s essence, fully consistent with the findings that many important biological functions of proteins can be elucidated with their low-frequency internal motions. To maximize the convenience of most experimental scientists, we have provided a step-by-step guide on how to use the predictor’s web server (http://www.jci-bioinfo.cn/iPPBS-Opt to get the desired results without the need to go through the complicated mathematical equations involved.

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

    Directory of Open Access Journals (Sweden)

    Piyaporn Phansak

    2016-06-01

    Full Text Available 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 R2 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.

  4. A multi-isotope ({delta}D, {delta}{sup 18}O, {sup 87}Sr/{sup 86}Sr, and {delta}{sup 11}B) approach for identifying saltwater intrusion and resolving groundwater evolution along the Western Caprock Escarpment of the Southern High Plains, New Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Langman, Jeff B., E-mail: jblangman@miners.utep.edu [University of Texas at El Paso, Geological Sciences, El Paso, TX 79968-0555 (United States); Ellis, Andre S. [University of Texas at El Paso, Geological Sciences, El Paso, TX 79968-0555 (United States)

    2010-01-15

    Declining water levels in arid and semi-arid regions increase an aquifer's vulnerability to natural and anthropogenic influences. A multi-isotope ({delta}D, {delta}{sup 18}O, {sup 87}Sr/{sup 86}Sr, and {delta}{sup 11}B) approach was used to resolve the geochemical evolution of groundwater in a declining aquifer in a semi-arid region of the southwestern USA as groundwater composition reacts to source-water mixing, cross-formational flow including saltwater intrusion, water-rock interaction, and likely agricultural recharge. Sub-aquifers or local flow systems are present in the Southern High Plains aquifer along the Western Caprock Escarpment in New Mexico, and the study site's local flow system contains a Na-Cl, high dissolved-solids groundwater that flows from the escarpment until it mixes with a high quality regional aquifer or regional flow system. The local flow system contains water that is similar in composition to the underlying, upper Dockum Group aquifer. Saltwater found in the upper Dockum Group aquifer likely originates in the adjacent Pecos River Basin and crosses beneath or possibly through the hydrologic divide of the Western Caprock Escarpment. Strontium concentrations of 0.9-31 mg/L and a {sup 87}Sr/{sup 86}Sr range of 0.70845-0.70906 were sufficient to estimate source-water fractions, mixing patterns, and contributions from chemical weathering through mass balance inverse calculations. Boron concentrations (59-1740 mg/L) and {delta}{sup 11}B values (+6.0-+46.0 per mille) were used to confirm source-water mixing, further evaluate water-rock interaction, and examine the influence of possible agricultural recharge. Alteration of B concentrations and {delta}{sup 11}B values in an area of likely agricultural recharge indicated the loss of B and decrease in {delta}{sup 11}B values likely from plant uptake, adsorption, and weathering contributions in the soil/vadose zone prior to recharge. The effectiveness of {sup 87}Sr/{sup 86}Sr and {delta

  5. 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

  6. 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.

  7. A quantitative proteomic approach to identify significantly altered protein networks in the serum of patients with lymphangioleiomyomatosis (LAM.

    Directory of Open Access Journals (Sweden)

    Nessa Banville

    Full Text Available Lymphangioleiomyomatosis (LAM is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05. Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, p = 0.03, von Willebrand Factor (40% reduction in LAM, p = 0.03 and Kallikrein III (25% increase in LAM, p = 0.03. Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future.

  8. 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...... by the two cell lines. The study demonstrates a quantitative and comparative proteomic strategy to identify clinically-relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy....

  9. 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...

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

    Science.gov (United States)

    Damgaard, Christian; Jensen, Lars J.; Holmstrup, Palle

    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 with LysC and trypsin. The resulting peptide mixtures were cleaned up by solid-phase extraction and separated online with 2 h gradients by nano-scale C18 reversed-phase chromatography connected to a mass spectrometer through an electrospray source. The eluting peptides were analyzed on a tandem mass spectrometer operated in data-dependent acquisition mode. Results We identified a total of 35,664 unique peptides from 4,161 different proteins, of which 1,946 and 2,090 were of bacterial and human origin, respectively. The human protein profiles displayed significant overexpression of the complement system 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 status. Similar bacterial proteomes in healthy and diseased individuals suggests that the salivary microbiota predominantly thrives in a planktonic state expressing no disease-associated characteristics of metabolic activity. PMID:27672500

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

    Directory of Open Access Journals (Sweden)

    Daniel Belstrøm

    2016-09-01

    Full Text Available 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 with LysC and trypsin. The resulting peptide mixtures were cleaned up by solid-phase extraction and separated online with 2 h gradients by nano-scale C18 reversed-phase chromatography connected to a mass spectrometer through an electrospray source. The eluting peptides were analyzed on a tandem mass spectrometer operated in data-dependent acquisition mode. Results We identified a total of 35,664 unique peptides from 4,161 different proteins, of which 1,946 and 2,090 were of bacterial and human origin, respectively. The human protein profiles displayed significant overexpression of the complement system 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 status. Similar bacterial proteomes in healthy and diseased individuals suggests that the salivary microbiota predominantly thrives in a planktonic state expressing no disease-associated characteristics of metabolic activity.

  12. In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9

    Directory of Open Access Journals (Sweden)

    Jing Mi

    2016-01-01

    Full Text Available The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.

  13. HmCRIP, a cysteine-rich intestinal protein, is expressed by an identified regenerating nerve cell.

    Science.gov (United States)

    Emes, R D; Wang, W-Z; Lanary, K; Blackshaw, S E

    2003-01-02

    A Hirudo medicinalis cDNA isolated from regenerating CNS tissue at 24 h post-axotomy was identified as a leech homologue of the mammalian cysteine-rich intestinal proteins (CRIPs) and named HmCRIP. HmCRIP is up-regulated within 6 h of axotomy, peaking at 24 h. This is the first demonstration of a CRIP homologue in regenerating CNS and in a serotonergic neurone. In rodents CRIP is an important factor in the regulation of the inflammatory immune response through control of Th1/Th2 differentiation. The role of HmCRIP in the regeneration competent environment of the annelid central nervous system is discussed.

  14. Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA.

    Science.gov (United States)

    Cho, Sung Ki; Kang, Il-Ho; Carr, Tyrell; Hannapel, David J

    2012-01-01

    Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3' untranslated region (UTR) of the RNA in mediating transport. Because the 3' UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3' UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3' UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

  15. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  16. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

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    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  17. Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

    Science.gov (United States)

    Tang, W X; Bazaraa, H M; Magiera, H; Cooke, N E; Haddad, J G

    1996-06-01

    Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

  18. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

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    Gabriel Gomez

    Full Text Available Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  19. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

    Science.gov (United States)

    Gomez, Gabriel; Pei, Jianwu; Mwangi, Waithaka; Adams, L Garry; Rice-Ficht, Allison; Ficht, Thomas A

    2013-01-01

    Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  20. Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

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    Kosuke Tsukamoto

    Full Text Available BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293 for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

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

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    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.

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

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    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.

  3. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    Energy Technology Data Exchange (ETDEWEB)

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  4. Identifying placental epigenetic alterations in an intrauterine growth restriction (IUGR) rat model induced by gestational protein deficiency.

    Science.gov (United States)

    Reamon-Buettner, Stella Marie; Buschmann, Jochen; Lewin, Geertje

    2014-06-01

    Poor maternal nutrition during gestation can lead to intrauterine growth retardation (IUGR), a main cause of low birth weight associated with high neonatal morbidity and mortality. Such early uterine environmental exposures can impact the neonatal epigenome to render later-in-life disease susceptibility. We established in Wistar Han rats a mild IUGR model induced by gestational protein deficiency (i.e. 9% crude protein in low protein diet vs. 21% in control, from GD 0 to 21) to identify alterations in gene expression and methylation patterns in certain genes implicated in human IUGR or in placental development. We found differential gene expression of Wnt2 and Dlk1 between IUGR and control. Notably, Wnt2 exhibited significant decrease while Dlk1 increase in IUGR placentas, correlating to decrease in fetal and placental weight. Methylation patterns encompassing 30 CpGs in the Wnt2 promoter region revealed variability in both IUGR and control placentas, but a site-specific hypomethylation was evident in IUGR placentas. Our present findings further support a key role of maternal gestational nutrition in defining the neonatal epigenome. Copyright © 2014. Published by Elsevier Inc.

  5. Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen

    Science.gov (United States)

    Lakhssassi, Naoufal; Liu, Shiming; Bekal, Sadia; Zhou, Zhou; Colantonio, Vincent; Lambert, Kris; Barakat, Abdelali; Meksem, Khalid

    2017-01-01

    Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN. PMID:28338077

  6. Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

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    Shakes Leighcraft A

    2012-09-01

    Full Text Available Abstract Background Non-coding DNA in and around the human Amyloid Precursor Protein (APP gene that is central to Alzheimer’s disease (AD shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. Results Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription

  7. A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response.

    Science.gov (United States)

    Zhang, Qiong; Song, Xueying; Su, Peng; Li, Ranran; Liu, Chang; Gou, Meng; Wang, Hao; Liu, Xin; Li, Qingwei

    2016-04-01

    The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named "Lja-Fyn". The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family.

  8. Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins.

    Science.gov (United States)

    Stevens, M R; Coleman, C E; Parkinson, S E; Maughan, P J; Zhang, H-B; Balzotti, M R; Kooyman, D L; Arumuganathan, K; Bonifacio, A; Fairbanks, D J; Jellen, E N; Stevens, J J

    2006-05-01

    Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0x coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.

  9. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

    Science.gov (United States)

    Voter, Andrew F; Manthei, Kelly A; Keck, James L

    2016-07-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

  10. Comparative analysis of Mafriwal (Bos taurus × Bos indicus) and Kedah Kelantan (Bos indicus) sperm proteome identifies sperm proteins potentially responsible for higher fertility in a tropical climate.

    Science.gov (United States)

    Ashrafzadeh, Ali; Nathan, Sheila; Karsani, Saiful Anuar

    2013-07-30

    The fertility of zebu cattle (Bos indicus) is higher than that of the European purebred (Bos taurus) and crossbred (Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility.

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

    Science.gov (United States)

    Brownjohn, Philip W; Smith, James; Portelius, Erik; Serneels, Lutgarde; Kvartsberg, Hlin; De Strooper, Bart; Blennow, Kaj; Zetterberg, Henrik; Livesey, Frederick J

    2017-04-11

    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. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. X-ray instrumentation for SR beamlines

    CERN Document Server

    Kovalchuk, M V; Zheludeva, S I; Aleshko-Ozhevsky, O P; Arutynyan, E H; Kheiker, D M; Kreines, A Y; Lider, V V; Pashaev, E M; Shilina, N Y; Shishkov, V A

    2000-01-01

    The main possibilities and parameters of experimental X-ray stations are presented: 'Protein crystallography', 'X-ray structure analysis', 'High-precision X-ray optics', 'X-ray crystallography and material science', 'X-ray topography', 'Photoelectron X-ray standing wave' that are being installed at Kurchatov SR source by A.V. Shubnikov Institute of Crystallography.

  13. ROC and confusion analysis of structure comparison methods identify the main causes of divergence from manual protein classification

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    Gibrat Jean-Francois

    2006-04-01

    Full Text Available Abstract Background Current classification of protein folds are based, ultimately, on visual inspection of similarities. Previous attempts to use computerized structure comparison methods show only partial agreement with curated databases, but have failed to provide detailed statistical and structural analysis of the causes of these divergences. Results We construct a map of similarities/dissimilarities among manually defined protein folds, using a score cutoff value determined by means of the Receiver Operating Characteristics curve. It identifies folds which appear to overlap or to be "confused" with each other by two distinct similarity measures. It also identifies folds which appear inhomogeneous in that they contain apparently dissimilar domains, as measured by both similarity measures. At a low (1% false positive rate, 25 to 38% of domain pairs in the same SCOP folds do not appear similar. Our results suggest either that some of these folds are defined using criteria other than purely structural consideration or that the similarity measures used do not recognize some relevant aspects of structural similarity in certain cases. Specifically, variations of the "common core" of some folds are severe enough to defeat attempts to automatically detect structural similarity and/or to lead to false detection of similarity between domains in distinct folds. Structures in some folds vary greatly in size because they contain varying numbers of a repeating unit, while similarity scores are quite sensitive to size differences. Structures in different folds may contain similar substructures, which produce false positives. Finally, the common core within a structure may be too small relative to the entire structure, to be recognized as the basis of similarity to another. Conclusion A detailed analysis of the entire available protein fold space by two automated similarity methods reveals the extent and the nature of the divergence between the automatically

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Anne Poinsignon

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  16. A novel protein-coding ORF72.2 gene was identified from Marek's disease virus strain CVI988

    Directory of Open Access Journals (Sweden)

    Liu Ping

    2010-12-01

    Full Text Available Abstract Marek's disease is a highly contagious disease of poultry characterized by rapid-on set of T-cell lymphomas, which is caused by Marek's disease virus (MDV, but its pathogenic mechanism is still not very clear. Recently, some new progress were achieved in molecular character of MDV. Along with the genomic sequencing of MDV serotype 1, some novel open reading frames (ORFs were predicted, and ORF72.2 was one of them which have no homologues in other MDV serotypes or in other alphaherpesvirus. In the study, ORF72.2 was firstly identified as a protein-coding gene by the method of reverse transcription polymerase chain reaction (RT-PCR, western blotting and indirect immunofluorescence assay. This study paved the way to conduct further studies to determine whether ORF72.2 plays a role in MDV replication and pathogenicity.

  17. Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Paola Checconi

    Full Text Available Protein cysteines can form transient disulfides with glutathione (GSH, resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX 1, PRDX2, vimentin (VIM, profilin1 (PFN1 and thioredoxin 1 (TXN1. For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX, which also inhibited tumor necrosis factor (TNF-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC, which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

  18. An identified antioxidant peptide obtained from ostrich (Struthio camelus) egg white protein hydrolysate shows wound healing properties.

    Science.gov (United States)

    Homayouni-Tabrizi, Masoud; Asoodeh, Ahmad; Abbaszadegan, Mohammad-Reza; Shahrokhabadi, Khadijeh; Nakhaie Moghaddam, Mahboobeh

    2015-08-01

    Ostrich (Struthio camelus) egg possesses a high amount of food proteins and thus plays an important role in nutrition. Ostrich egg white proteins were hydrolyzed with pepsin and pancreatin to examine its antioxidant properties and further characterized the most active peptide. Ostrich egg white protein hydrolysate (OEWPH) was fractionized using reversed phase high-pressure liquid chromatography (HPLC). The antioxidant activity of OEWPH and its HPLC fraction were investigated based on their scavenging capacity1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), superoxide ([Formula: see text]), hydroxyl (OH(•-)) radicals, and Cu(+2) chelating. In a wound healing assay, paravertebral excision (1 cm diameter) was made on the skin and the percentage of wound closure was measured at defined intervals (0, 3, 7, and 14 d). A potent antioxidant peptide named DG-10 with the sequence DAESLSRLLG (MW: 1060.18 ± 0.5 Da) was identified from OEWPH. The peptide DG-10 showed DPPH (IC50 = 0.0085 mg/ml), ABTS(•+) (IC50 = 0.56 mg/ml), superoxide (IC50 = 0.36 mg/ml), and hydroxyl (IC50 = 0.4 mg/ml) radical scavenger and copper chelating activity (IC50 = 0.28 mg/ml). In vitro cultured HFLF-pI 5, the cell model, also revealed that DG-10 could protect HFLF-pI 5 cells against H2O2-treated necrosis. Ointment composed of DG-10 peptide exhibited wound-healing properties on adult rats (Wistar strain). The percentage of wound closure in peptide-treated group was 98% by day 14. Our results suggested that DG-10 is a natural agent obtained from ostrich egg possessing considerable antioxidant and wound-healing properties.

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

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    Anne N Shemon

    Full Text Available BACKGROUND: 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. METHODS/FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  20. Identifying Metabolite and Protein Biomarkers in Unstable Angina In-patients by Feature Selection Based Data Mining Method

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng-he; YANG Yi; WANG Wei; ZHAO Hui-hui; HOU Na; CHEN Jian-xin; SHI Qi; XU Xue-gong; WANG Juan; ZHENG Cheng-long; ZHAO Ling-yan

    2011-01-01

    Unstable angina(UA) is the most dangerous type of Coronary Heart Disease(CHD) to cause more and more mortal and morbid world wide. Identification of biomarkers for UA at the level of proteomics and metaboiomics is a better avenue to understand the inner mechanism of it. Feature selection based data mining method is better suited to identify biomarkers of UA. In this study, we carried out clinical epidemiology to collect plasmas of UA in-patients and controls. Proteomics and metabolomics data were obtained via two-dimensional difference gel electrophoresis and gas chromatography techniques. We presented a novel computational strategy to select biomarkers as few as possible for UA in the two groups of data. Firstly, decision tree was used to select biomarkers for UA and 3-fold cross validation was used to evaluate computational performances for the three methods. Alternatively, we combined independent t test and classification based data mining method as well as backward elimination technique to select, as few as possible, protein and metabolite biomarkers with best classification performances. By the method, we selected 6 proteins and 5 metabolites for UA. The novel method presented here provides a better insight into the pathology of a disease.

  1. RNA sequencing and pathway analysis identify tumor necrosis factor alpha driven small proline-rich protein dysregulation in chronic rhinosinusitis.

    Science.gov (United States)

    Ramakrishnan, Vijay R; Gonzalez, Joseph R; Cooper, Sarah E; Barham, Henry P; Anderson, Catherine B; Larson, Eric D; Cool, Carlyne D; Diller, John D; Jones, Kenneth; Kinnamon, Sue C

    2017-09-01

    Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder in which many pathways contribute to end-organ disease. Small proline-rich proteins (SPRR) are polypeptides that have recently been shown to contribute to epithelial biomechanical properties relevant in T-helper type 2 inflammation. There is evidence that genetic polymorphism in SPRR genes may predict the development of asthma in children with atopy and, correlatively, that expression of SPRRs is increased under allergic conditions, which leads to epithelial barrier dysfunction in atopic disease. RNAs from uncinate tissue specimens from patients with CRS and control subjects were compared by RNA sequencing by using Ingenuity Pathway Analysis (n = 4 each), and quantitative polymerase chain reaction (PCR) (n = 15). A separate cohort of archived sinus tissue was examined by immunohistochemistry (n = 19). A statistically significant increase of SPRR expression in CRS sinus tissue was identified that was not a result of atopic presence. SPRR1 and SPRR2A expressions were markedly increased in patients with CRS (p < 0.01) on RNA sequencing, with confirmation by using real-time PCR. Immunohistochemistry of archived surgical samples demonstrated staining of SPRR proteins within squamous epithelium of both groups. Pathway analysis indicated tumor necrosis factor (TNF) alpha as a master regulator of the SPRR gene products. Expression of SPRR1 and of SPRR2A is increased in mucosal samples from patients with CRS and appeared as a downstream result of TNF alpha modulation, which possibly resulted in epithelial barrier dysfunction.

  2. Panicovirus accumulation is governed by two membrane-associated proteins with a newly identified conserved motif that contributes to pathogenicity

    Directory of Open Access Journals (Sweden)

    Turina Massimo

    2006-03-01

    Full Text Available Abstract Panicum mosaic virus (PMV has a positive-sense, single-stranded RNA genome that serves as the mRNA for two 5'-proximal genes, p48 and p112. The p112 open reading frame (ORF has a GDD-motif, a feature of virus RNA-dependent RNA polymerases. Replication assays in protoplasts showed that p48 and p112 are sufficient for replication of PMV and its satellite virus (SPMV. Differential centrifugation of extracts from PMV-infected plants showed that the p48 and p112 proteins are membrane-associated. The same fractions exhibited RNA polymerase activity in vitro on viral RNA templates, suggesting that p48 and p112 represent the viral replication proteins. Moreover, we identified a domain spanning amino acids 306 to 405 on the p48 and p112 PMV ORFs that is common to the Tombusviridae. Alanine scanning mutagenesis of the conserved domain (CD revealed that several substitutions were lethal or severely debilitated PMV accumulation. Other substitutions did not affect RNA accumulation, yet they caused variable phenotypes suggestive of plant-dependent effects on systemic invasion and symptom induction. The mutants that were most debilitating to PMV replication were hydrophobic amino acids that we hypothesize are important for membrane localization and functional replicase activity.

  3. A Systematic Bioinformatics Approach to Identify High Quality Mass Spectrometry Data and Functionally Annotate Proteins and Proteomes.

    Science.gov (United States)

    Islam, Mohammad Tawhidul; Mohamedali, Abidali; Ahn, Seong Beom; Nawar, Ishmam; Baker, Mark S; Ranganathan, Shoba

    2017-01-01

    In the past decade, proteomics and mass spectrometry have taken tremendous strides forward, particularly in the life sciences, spurred on by rapid advances in technology resulting in generation and conglomeration of vast amounts of data. Though this has led to tremendous advancements in biology, the interpretation of the data poses serious challenges for many practitioners due to the immense size and complexity of the data. Furthermore, the lack of annotation means that a potential gold mine of relevant biological information may be hiding within this data. We present here a simple and intuitive workflow for the research community to investigate and mine this data, not only to extract relevant data but also to segregate usable, quality data to develop hypotheses for investigation and validation. We apply an MS evidence workflow for verifying peptides of proteins from one's own data as well as publicly available databases. We then integrate a suite of freely available bioinformatics analysis and annotation software tools to identify homologues and map putative functional signatures, gene ontology and biochemical pathways. We also provide an example of the functional annotation of missing proteins in human chromosome 7 data from the NeXtProt database, where no evidence is available at the proteomic, antibody, or structural levels. We give examples of protocols, tools and detailed flowcharts that can be extended or tailored to interpret and annotate the proteome of any novel organism.

  4. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Tandem reporter assay for myristoylated proteins post-translationally (TRAMPP) identifies novel substrates for post-translational myristoylation: PKCε, a case study.

    Science.gov (United States)

    Martin, Dale D O; Ahpin, Chrisselle Y; Heit, Ryan J; Perinpanayagam, Maneka A; Yap, Megan C; Veldhoen, Richard A; Goping, Ing Swie; Berthiaume, Luc G

    2012-01-01

    Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.

  7. Geosphere process report for the safety assessment SR-Site

    Energy Technology Data Exchange (ETDEWEB)

    Skagius, Kristina (ed.) (Kemakta Konsult AB, Stockholm (Sweden))

    2010-11-15

    This report documents geosphere processes identified as relevant to the long-term safety of a KBS-3 repository, and forms an important part of the reporting of the safety assessment SR-Site. The detailed assessment methodology, including the role of the process reports in the assessment, is described in the SR-Site Main report /SKB 2011/

  8. Highly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array.

    Directory of Open Access Journals (Sweden)

    Caroline May

    Full Text Available Amyotrophic lateral sclerosis (ALS, the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.

  9. C-reactive protein and the MASCC risk index identify high-risk patients with febrile neutropenia and hematologic neoplasms.

    Science.gov (United States)

    Combariza, Juan F; Lombana, Milton; Pino, Luis E; Arango, Marcos

    2015-04-01

    The objective of this study is to assess the prognostic usefulness of the Multinational Association of Supportive Care in Cancer (MASCC) risk score in association with the value of C-reactive protein (CRP) to identify high-risk patients with febrile neutropenia and hematologic neoplasms. A retrospective cohort study in which the MASCC score and the CRP values were used to assess the mortality risk at 30 days among patients with febrile neutropenia and hematologic malignancies was performed. Two hundred thiry-seven patients with febrile neutropenia were analyzed; the mortality rate within 30 days was 9 %. High-risk patients according to the MASCC score were significantly more likely to experience adverse outcomes, such as being transferred to the intensive care unit (RR 3.55; CI 95 % 2.73-6.62, p risk group identified by the MASCC score (HR 3.0; CI 95 % 1.12-13.54, p = 0.032) and the mean levels of CRP (HR 17; CI 95 % 2.21-136.48, p = 0.007) and survival. The survival rate within 30 days was 100 % for the patients with a low-risk MASCC score and a mean CRP less than 15 mg/dL. This rate was only 64 % for high-risk patients with a mean CRP greater than 15 mg/dL. The MASCC risk score combined with the mean CRP value successfully identifies patients with febrile neutropenia and hematological malignancies and a high risk of death.

  10. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

    OpenAIRE

    Sae Tanaka; Junko Tanaka; Yoshihiro Miwa; Horikawa, Daiki D.; Toshiaki Katayama; Kazuharu Arakawa; Atsushi Toyoda; Takeo Kubo; Takekazu Kunieda

    2015-01-01

    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 se...

  11. 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

  12. 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.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    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 and were

  13. 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

  14. 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.

  15. Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools.

    Science.gov (United States)

    Ladics, Gregory S; Selgrade, MaryJane K

    2009-08-01

    A safety assessment process exists for genetically engineered crops that includes the evaluation of the expressed protein for allergenic potential. The objectives of this evaluation are twofold: (1) to protect allergic consumers from exposure to known allergenic or cross-reactive proteins, and (2) protect the general population from risks associated with the introduction of genes encoding proteins that are likely to become food allergens. The first systematic approach to address these concerns was formulated by Metcalfe et al. [Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., and Fuchs, R.L. 1996. Assessment of the allergenic potential of foods from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36(5), 165-186.] and subsequently Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) [FAO/WHO, 2001. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology. January 22-25, 2001. Rome, Italy]. More recently, Codex [Codex Alimentarius Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity. pp. 47-60], noting that no single factor is recognized as an identifier for protein allergenicity, suggested a weight of evidence approach be conducted that takes into account a variety of factors and approaches for an overall assessment of allergenic potential. These various recommendations are based on what is known about allergens, including the history of exposure and safety of the gene(s) source; amino acid sequence identity to human allergens; stability to pepsin digestion in vitro; protein abundance in the crop and

  16. An experimentally based computer search identifies unstructured membrane-binding sites in proteins: application to class I myosins, PAKS, and CARMIL.

    Science.gov (United States)

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D

    2010-02-19

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.

  17. In-depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites.

    Science.gov (United States)

    Champagne, Antoine; Rischer, Heiko; Oksman-Caldentey, Kirsi-Marja; Boutry, Marc

    2012-12-01

    Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D-DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.

  18. Validation of a Non-Targeted LC-MS Approach for Identifying Ancient Proteins: Method Development on Bone to Improve Artifact Residue Analysis

    Directory of Open Access Journals (Sweden)

    Andrew Barker

    2015-09-01

    Full Text Available Identification of protein residues from prehistoric cooking pottery using mass spectrometry is challenging because proteins are removed from original tissues, are degraded from cooking, may be poorly preserved due to diagenesis, and occur in a palimpsest of exogenous soil proteins. In contrast, bone proteins are abundant and well preserved. This research is part of a larger method-development project for innovation and improvement of liquid chromatography – mass spectrometry analysis of protein residues from cooking pottery; here we validate the potential of our extraction and characterization approach via application to ancient bone proteins. Because of its preservation potential for proteins and given that our approach is destructive, ancient bone identified via skeletal morphology represents an appropriate verification target. Proteins were identified from zooarchaeological turkey (Meleagris gallopavo Linnaeus Phasianidae, rabbit (Lagomorpha, and squirrel (Sciuridae remains excavated from ancient pueblo archaeological sites in southwestern Colorado using a non-targeted LC-MS/MS approach. The data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD002440. Improvement of highly sensitive targeted LC-MS/MS approaches is an avenue for future method development related to the study of protein residues from artifacts such as stone tools and pottery.

  19. Genome-wide association and functional studies identify a role for matrix Gla protein in osteoarthritis of the hand.

    Science.gov (United States)

    Hollander, Wouter den; Boer, Cindy G; Hart, Deborah J; Yau, Michelle S; Ramos, Yolande F M; Metrustry, Sarah; Broer, Linda; Deelen, Joris; Cupples, L Adrienne; Rivadeneira, Fernando; Kloppenburg, Margreet; Peters, Marjolein; Spector, Tim D; Hofman, Albert; Slagboom, P Eline; Nelissen, Rob G H H; Uitterlinden, André G; Felson, David T; Valdes, Ana M; Meulenbelt, Ingrid; van Meurs, Joyce J B

    2017-08-30

    Osteoarthritis (OA) is the most common form of arthritis and the leading cause of disability in the elderly. Of all the joints, genetic predisposition is strongest for OA of the hand; however, only few genetic risk loci for hand OA have been identified. Our aim was to identify novel genes associated with hand OA and examine the underlying mechanism. We performed a genome-wide association study of a quantitative measure of hand OA in 12 784 individuals (discovery: 8743, replication: 4011). Genome-wide significant signals were followed up by analysing gene and allele-specific expression in a RNA sequencing dataset (n=96) of human articular cartilage. We found two significantly associated loci in the discovery set: at chr12 (p=3.5 × 10(-10)) near the matrix Gla protein (MGP) gene and at chr12 (p=6.1×10(-9)) near the CCDC91 gene. The DNA variant near the MGP gene was validated in three additional studies, which resulted in a highly significant association between the MGP variant and hand OA (rs4764133, Betameta=0.83, Pmeta=1.8*10(-15)). This variant is high linkage disequilibrium with a coding variant in MGP, a vitamin K-dependent inhibitor of cartilage calcification. Using RNA sequencing data from human primary cartilage tissue (n=96), we observed that the MGP RNA expression of the hand OA risk allele was significantly lowercompared with the MGP RNA expression of the reference allele (40.7%, p<5*10(-16)). Our results indicate that the association between the MGP variant and increased risk for hand OA is caused by a lower expression of MGP, which may increase the burden of hand OA by decreased inhibition of cartilage calcification. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  20. Protein Biomarkers Identify Patients Unlikely to Benefit from Primary Prevention ICDs: Findings from the PROSE-ICD Study

    Science.gov (United States)

    Cheng, Alan; Zhang, Yiyi; Blasco-Colmenares, Elena; Dalal, Darshan; Butcher, Barbara; Norgard, Sanaz; Eldadah, Zayd; Ellenbogen, Kenneth A.; Dickfeld, Timm; Spragg, David D.; Marine, Joseph E.; Guallar, Eliseo; Tomaselli, Gordon F.

    2015-01-01

    Background Primary prevention implantable cardioverter defibrillators (ICDs) reduce all-cause mortality but the benefits are heterogeneous. Current risk stratification based on left ventricular ejection fraction has limited discrimination power. We hypothesize that biomarkers for inflammation, neurohumoral activation and cardiac injury can predict appropriate shocks and all-cause mortality in patients with primary prevention ICDs. Methods and Results The Prospective Observational Study of Implantable Cardioverter Defibrillators (PROSe-ICD) enrolled 1,189 patients with systolic heart failure who underwent ICD implantation for primary prevention of sudden cardiac death. The primary endpoint was an ICD shock for adjudicated ventricular tachyarrhythmia. The secondary endpoint was all-cause mortality. After a median follow-up of 4.0 years, 137 subjects experienced an appropriate ICD shock and 343 participants died (incidence rates of 3.2 and 5.8 per 100 person-years, respectively). In multivariable adjusted models, higher interleukin-6 (IL-6) levels increased the risk of appropriate ICD shocks. In contrast, C-reactive protein, IL-6, tumor necrosis factor-α receptor II, pro-brain natriuretic peptide, and cardiac troponin T showed significant linear trends for increased risk of all-cause mortality across quartiles. A score combining these 5 biomarkers identified patients who were much more likely to die than to receive an appropriate shock from the ICD. Conclusions An increase in serum biomarkers of inflammation, neurohumoral activation and myocardial injury increased the risk for death but poorly predicted the likelihood of an ICD shock. These findings highlight the potential importance of serum-based biomarkers in identifying patients who are unlikely to benefit from primary prevention ICDs. PMID:25273351

  1. A Novel Analytical Strategy to Identify Fusion Transcripts between Repetitive Elements and Protein Coding-Exons Using RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Tianyuan Wang

    Full Text Available Repetitive elements (REs comprise 40-60% of the mammalian genome and have been shown to epigenetically influence the expression of genes through the formation of fusion transcript (FTs. We previously showed that an intracisternal A particle forms an FT with the agouti gene in mice, causing obesity/type 2 diabetes. To determine the frequency of FTs genome-wide, we developed a TopHat-Fusion-based analytical pipeline to identify FTs with high specificity. We applied it to an RNA-seq dataset from the nucleus accumbens (NAc of mice repeatedly exposed to cocaine. Cocaine was previously shown to increase the expression of certain REs in this brain region. Using this pipeline that can be applied to single- or paired-end reads, we identified 438 genes expressing 813 different FTs in the NAc. Although all types of studied repeats were present in FTs, simple sequence repeats were underrepresented. Most importantly, reverse-transcription and quantitative PCR validated the expression of selected FTs in an independent cohort of animals, which also revealed that some FTs are the prominent isoforms expressed in the NAc by some genes. In other RNA-seq datasets, developmental expression as well as tissue specificity of some FTs differed from their corresponding non-fusion counterparts. Finally, in silico analysis predicted changes in the structure of proteins encoded by some FTs, potentially resulting in gain or loss of function. Collectively, these results indicate the robustness of our pipeline in detecting these new isoforms of genes, which we believe provides a valuable tool to aid in better understanding the broad role of REs in mammalian cellular biology.

  2. Identifying potential selective fluorescent probes for cancer-associated protein carbonic anhydrase IX using a computational approach.

    Science.gov (United States)

    Kamstra, Rhiannon L; Floriano, Wely B

    2014-11-01

    Carbonic anhydrase IX (CAIX) is a biomarker for tumor hypoxia. Fluorescent inhibitors of CAIX have been used to study hypoxic tumor cell lines. However, these inhibitor-based fluorescent probes may have a therapeutic effect that is not appropriate for monitoring treatment efficacy. In the search for novel fluorescent probes that are not based on known inhibitors, a database of 20,860 fluorescent compounds was virtually screened against CAIX using hierarchical virtual ligand screening (HierVLS). The screening database contained 14,862 compounds tagged with the ATTO680 fluorophore plus an additional 5998 intrinsically fluorescent compounds. Overall ranking of compounds to identify hit molecular probe candidates utilized a principal component analysis (PCA) approach. Four potential binding sites, including the catalytic site, were identified within the structure of the protein and targeted for virtual screening. Available sequence information for 23 carbonic anhydrase isoforms was used to prioritize the four sites based on the estimated "uniqueness" of each site in CAIX relative to the other isoforms. A database of 32 known inhibitors and 478 decoy compounds was used to validate the methodology. A receiver-operating characteristic (ROC) analysis using the first principal component (PC1) as predictive score for the validation database yielded an area under the curve (AUC) of 0.92. AUC is interpreted as the probability that a binder will have a better score than a non-binder. The use of first component analysis of binding energies for multiple sites is a novel approach for hit selection. The very high prediction power for this approach increases confidence in the outcome from the fluorescent library screening. Ten of the top scoring candidates for isoform-selective putative binding sites are suggested for future testing as fluorescent molecular probe candidates.

  3. SR study of scintillators

    CERN Document Server

    Mikhailin, V V

    2000-01-01

    The technique and the models developed recently in Synchrotron Radiation (SR) Laboratory of Moscow University for the investigation of energy transfer processes in insulators can be applied for a detailed study of scintillator's properties. The experiments with SR in VUV and XUV regions allow to separate various processes and stages of energy transfer and help to indicate the dominating process on each stage. Thus, we can find the way to improve the properties, essential for the particular application of the scintillator: conversion efficiency, decay time, afterglow, radiation hardness, etc. The results of such a study can be used for the testing procedures in scintillator production.

  4. SR 97 - Identification and structuring of process

    Energy Technology Data Exchange (ETDEWEB)

    Pers, K.; Skagius, K.; Soedergren, S.; Wiborgh, M. [Kemakta Konsult AB, Stockholm (Sweden); Hedin, A.; Moren, L.; Sellin, P.; Stroem, A. [Swedish Nuclear Fuel and Waste Management Co., Stockholm (Sweden); Pusch, R. [Geodevelopment AB, Lund (Sweden); Bruno, J. [QuantiSci SL, Barcelona (Spain)

    1999-12-01

    This report documents work conducted in recent years to identify processes and interactions of importance to the evaluation of long-term safety of a KBS 3 type deep repository for spent nuclear fuel. Previous, partly undocumented work regarding interaction matrices is described as well as the THMC diagrams that have been used in the safety assessment SR 97. The coupling between the two sources of information is documented in a database. In the same database, the interaction matrices are briefly documented, while the processes in the THMC diagrams are more thoroughly documented in a special so called Process Report, which forms an important supporting document for SR 97.

  5. Nature and Significance of the High-Sr Aleutian Lavas

    Science.gov (United States)

    Yogodzinski, G. M.; Arndt, S.; Turka, J. R.; Kelemen, P. B.; Vervoort, J. D.; Portnyagin, M.; Hoernle, K.

    2011-12-01

    Results of the Western Aleutian Volcano Expedition and German-Russian KALMAR cruises include the discovery of seafloor volcanism at the Ingenstrem Depression and at unnamed seamounts 300 km west of Buldir, the westernmost emergent volcano in the Aleutian arc. These discoveries indicate that the surface expression of active Aleutian volcanism goes below sea level just west of Buldir, but is otherwise continuous along the full length of the arc. Many lavas dredged from western Aleutian seamounts are basalts, geochemically similar to basalts from elsewhere in Aleutians and other arcs (La/Yb 4-8, Sr/Y700 ppm Sr), which are mostly plagioclase-hornblende andesites and dacites with low Y and middle-heavy rare-earth elements, fractionated trace element patterns (Sr/Y=50-200, La/Yb=9-25) and MORB-like isotopes (87Sr/86Sr 0.65) with 1250-1700 ppm Sr, 4-7 ppm Y, low abundances of all rare-earth elements (Laandesites from some emergent volcanoes in the western Aleutians, and mixing arrays indicate that it may be present in all Aleutian lavas (e.g., 87Sr/86Sr vs. La/Yb or Sr/Y); however, radiogenic Pb and Sr from subducted sediment renders the high-Sr endmember isotopically invisible in most central and eastern Aleutian lavas. The geochemistry of small monogenetic sea-floor volcanoes--especially those in the back-arc--may be the best opportunity to identify the high-Sr endmember in central and eastern Aleutian locations. The existence of primitive, high-silica lavas in the western Aleutians, where the subducting plate is probably unusually hot, may also provide key observations toward an improved understanding of high-Mg# andesites and dacites from other hot-slab locations, especially in the Cascades and Central Mexico. [1] Zimmer et al., 2010, J. Petrology, v. 51, p. 2411

  6. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    Science.gov (United States)

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  7. Phage-display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential

    Science.gov (United States)

    The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell recepto...

  8. Targeting SR-BI for cancer diagnostics, imaging and therapy

    Directory of Open Access Journals (Sweden)

    Maneesha Amrita Rajora

    2016-09-01

    Full Text Available Scavenger receptor class B type I (SR-BI plays an important role in trafficking cholesteryl esters between the core of high density lipoprotein and the liver. Interestingly, this integral membrane protein receptor is also implicated in the metabolism of cholesterol by cancer cells, whereby overexpression of SR-BI has been observed in a number of tumours and cancer cell lines, including breast and prostate cancers. Consequently, SR-BI has recently gained attention as a cancer biomarker and exciting target for the direct cytosolic delivery of therapeutic agents. This brief review highlights these key developments in SR-BI-targeted cancer therapies and imaging probes. Special attention is given to the exploration of high density lipoprotein nanomimetic platforms that take advantage of upregulated SR-BI expression to facilitate targeted drug-delivery and cancer diagnostics, and promising future directions in the development of these agents.

  9. 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

    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...... origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r...

  10. Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy

    Directory of Open Access Journals (Sweden)

    Dobson Wendy

    2011-06-01

    Full Text Available Abstract Background RNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK family of SR protein kinases on HIV-1 expression and RNA processing was examined. Results Despite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication. Conclusions Together, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies.

  11. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    Directory of Open Access Journals (Sweden)

    Imhof Axel

    2007-07-01

    Full Text Available Abstract Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra.

  12. The skeletal proteome of the brittle star Ophiothrix spiculata identifies C-type lectins and other proteins conserved in echinoderm skeleton formation

    Directory of Open Access Journals (Sweden)

    Brian T. Livingston

    2016-07-01

    Full Text Available Determining the identity and functional role of proteins involved in biomineralization and the formation of skeletons is critical to our understanding of the process. Proteomics has allowed rapid characterization of the proteins occluded within mineralized tissue, but the large numbers of proteins detected makes it difficult to assign the relative importance of each protein. We have taken a comparative approach, examining the skeletal proteome of different species of echinoderms in order to identify the proteins that are conserved and likely to be important. Our previous study comparing the skeletal proteome of the brittle star Ophiocoma wendtii to the published proteomes of the sea urchin Strongylocentrotus purpuratus revealed some conservation of proteins, but indicated that the C-type lectin domain-containing spicule matrix proteins abundant in the sea urchin skeletal proteome were not conserved in the brittle star. Here we examine the skeletal proteome of a different species of brittle star, Ophiothrix spiculata. We have isolated the proteins from the skeleton of O. spiculata and performed LC/MS/MS to identify peptides present. Comparison to transcriptome and genome databases revealed the proteins present in the O. spiculata proteome. Despite being diverged for several million years, the two brittle stars have very similar proteins in their skeletons. Included is a fibrinogen C-like lectin and several C-type lectins proteins, which we describe in detail. The unusual number of C-type lectins found in the S. purpuatus skeleton and the repetitive regions seen in those spicule matrix proteins are not present in O. spiculata.

  13. Novel biomarkers of nasopharyngeal carcinoma metastasis risk identified by reverse phase protein array based tumor profiling with consideration of plasma Epstein-Barr virus DNA load.

    Science.gov (United States)

    Xu, Tao; Su, Bojin; Huang, Peiyu; Wei, Weihong; Deng, Yanming; Sehgal, Vasudha; Wang, Donghui; Jiang, Jun; Zhang, Guoyi; Li, Anfei; Yang, Huiling; Claret, Francois X

    2017-05-01

    In patients with Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC), intertumor heterogeneity causes interpatient heterogeneity in the risk of distant metastasis. We aimed to identify novel biomarkers of metastasis risk using reverse phase protein array (RPPA) profiling of NPC patients at risk for metastasis and considering plasma EBV DNA load. A total of 98 patients with NPC with and without metastasis after treatment, matched with respect to clinical parameters, are enrolled. Total protein expression is measured by RPPA, and protein functions are analyzed by pathway bioinformatics. The RPPA analysis revealed a profile of 70 proteins that are differentially expressed in metastatic and nonmetastatic tumors. Plasma EBV DNA load after treatment correlated with protein expression level better than plasma EBV DNA load before treatment did. The biomarkers of NPC metastasis identified by proteomics regulate signaling pathways involved in cell cycle progression, apoptosis, and epithelial-mesenchymal transition. The authors identified 26 biomarkers associated with 5-year distant failure-free survival in univariate analysis; five biomarkers remained significant in multivariate analysis. A comprehensive RPPA profiling study is warranted to identify novel metastasis-related biomarkers and further examine the activation state of signaling proteins to improve estimation of metastasis risk for patients with EBV-associated NPC. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 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.

  15. The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages.

    Science.gov (United States)

    Asha, Srinivasan; Soniya, E V

    2017-02-01

    Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5' end of the putative long form of 5.8S rRNA (5.8SLrRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5' consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5'5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5'5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants.

  16. A set of host proteins interacting with papaya ringspot virus NIa-Pro protein identified in a yeast two-hybrid system.

    Science.gov (United States)

    Gao, L; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2012-01-01

    The protein-protein interactions between viral and host proteins play an essential role in plant virus infection and host defense. The potyviral nuclear inclusion protein a protease (NIa-Pro) is involved in various steps of viral infection. In this study, the host proteins interacting with papaya ringspot virus (PRSV) NIa-Pro were screened in a Carica papaya L. plant cDNA library using a Sos recruitment two-hybrid system (SRS). We confirmed that the full-length EIF3G, FBPA1, FK506BP, GTPBP, MSRB1, and MTL from papaya can interact specifically with PRSV NIa-Pro in yeast, respectively. These proteins fufill important functions in plant protein translation, biotic and abiotic stress, energy metabolism and signal transduction. In this paper, we discuss possible functions of interactions between these host proteins and NIa-Pro in PRSV infection and their role in host defense. Sos recruitment two-hybrid system; papaya ringspot virus; NIa-Pro; protein-protein interaction.

  17. Proteome analysis. Novel proteins identified at the peribacteroid membrane from ¤Lotus japonicus¤ root nodules

    DEFF Research Database (Denmark)

    Wienkoop, S.; Saalbach, G.

    2003-01-01

    The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two...... physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM....

  18. 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.

  19. Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

    Science.gov (United States)

    Kanugula, Anantha Koteswararao; Dhople, Vishnu M; Völker, Uwe; Ummanni, Ramesh; Kotamraju, Srigiridhar

    2014-01-01

    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.

  20. 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

    2017-06-05

    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 neighbours of a query protein based on six 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 homologues and orthologues. This article is protected by copyright. All rights reserved. © 2017 The Protein Society.

  1. SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein.

    Science.gov (United States)

    Sakinc, Türkan; Kleine, Britta; Gatermann, Sören G

    2006-08-01

    A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

  2. SR-71 flyover

    Science.gov (United States)

    1990-01-01

    This clip, running about 14 seconds in length, shows the NASA SR-71 (No. 844) lighting off the afterburners on a low pass over the Dryden Flight Research Center. Two SR-71A aircraft on loan from the U.S. Air Force have been used for high-speed, high-altitude research at the NASA Dryden Flight Research Center, Edwards, California, since 1991. One of them was later returned to the Air Force. A third SR-71 on loan from the Air Force is an SR-71B used for training but not for flight research. Developed for the U.S. Air Force as reconnaissance aircraft more than 30 years ago, SR-71 aircraft are still the world's fastest and highest-flying production aircraft. These aircraft can fly more than 2200 miles per hour (Mach 3+ or more than three times the speed of sound) and at altitudes of over 85,000 feet. This operating environment makes the aircraft excellent platforms to carry out research and experiments in a variety of areas--aerodynamics, propulsion, structures, thermal protection materials, high-speed and high-temperature instrumentation, atmospheric studies, and sonic-boom characterization. Data from the SR-71 high-speed research program may be used to aid designers of future supersonic or hypersonic aircraft and propulsion systems, including a possible high-speed civil transport. The SR-71 program at Dryden has been part of the NASA overall high-speed aeronautical research program, and projects have involved other NASA research centers, other government agencies, universities, and commercial firms. One of the first major experiments to be flown in the NASA SR-71 program was a laser air-data collection system. This system used laser light instead of air pressure to produce airspeed and attitude reference data such as angle of attack and angle of sideslip. These data are normally obtained with small tubes and vanes extending into the air stream, or from tubes with flush openings on the aircraft outer skin. The flights provided information on the presence of

  3. SR-71 flight

    Science.gov (United States)

    1990-01-01

    The movie clip shown here runs about 13 seconds and shows an air-to-air shot of the front of the SR-71 aircraft and a head-on view of it coming in for a landing. Two SR-71A aircraft on loan from the U.S. Air Force have been used for high-speed, high-altitude research at the NASA Dryden Flight Research Center, Edwards, California, since 1991. One of them was later returned to the Air Force. A third SR-71 on loan from the Air Force is an SR-71B used for training but not for flight research. Developed for the U.S. Air Force as reconnaissance aircraft more than 30 years ago, SR-71 aircraft are still the world's fastest and highest-flying production aircraft. These aircraft can fly more than 2200 miles per hour (Mach 3+ or more than three times the speed of sound) and at altitudes of over 85,000 feet. This operating environment makes the aircraft excellent platforms to carry out research and experiments in a variety of areas--aerodynamics, propulsion, structures, thermal protection materials, high-speed and high-temperature instrumentation, atmospheric studies, and sonic-boom characterization. Data from the SR-71 high-speed research program may be used to aid designers of future supersonic or hypersonic aircraft and propulsion systems, including a possible high-speed civil transport. The SR-71 program at Dryden has been part of the NASA overall high-speed aeronautical research program, and projects have involved other NASA research centers, other government agencies, universities, and commercial firms. One of the first major experiments to be flown in the NASA SR-71 program was a laser air-data collection system. This system used laser light instead of air pressure to produce airspeed and attitude reference data such as angle of attack and angle of sideslip. These data are normally obtained with small tubes and vanes extending into the air stream, or from tubes with flush openings on the aircraft outer skin. The flights provided information on the presence of

  4. The Yeast Split-Ubiquitin Membrane Protein Two-Hybrid Screen Identifies BAP31 as a Regulator of the Turnover of Endoplasmic Reticulum-Associated Protein Tyrosine Phosphatase-Like B

    OpenAIRE

    Wang, Bing; Pelletier, Jerry; Massaad, Michel J.; Herscovics, Annette; Shore, Gordon C

    2004-01-01

    In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. He...

  5. CaCO3 and SrCO3 bioprecipitation by fungi isolated from calcareous soil.

    Science.gov (United States)

    Li, Qianwei; Csetenyi, Laszlo; Paton, Graeme Iain; Gadd, Geoffrey Michael

    2015-08-01

    The urease-positive fungi Pestalotiopsis sp. and Myrothecium gramineum, isolated from calcareous soil, were examined for their properties of CaCO3 and SrCO3 biomineralization. After incubation in media amended with urea and CaCl2 and/or SrCl2 , calcite (CaCO3 ), strontianite (SrCO3 ), vaterite in different forms [CaCO3 , (Cax Sr1-x )CO3 ] and olekminskite [Sr(Sr,Ca)(CO3 )2 ] were precipitated, and fungal 'footprints' were observed on mineral surfaces. The amorphous precipitate mediated by Pestalotiopsis sp. grown with urea and equivalent concentrations of CaCl2 and SrCl2 was identified as hydrated Ca and Sr carbonates by Fourier transform infrared spectroscopy. Liquid media experiments showed M. gramineum possessed the highest Sr(2+) removal ability, and ∼ 49% of supplied Sr(2+) was removed from solution when grown in media amended with urea and 50 mM SrCl2 . Furthermore, this organism could also precipitate 56% of the available Ca(2+) and 28% of the Sr(2+) in the form of CaCO3 , SrCO3 and (Cax Sr1-x )CO3 when incubated in urea-amended media and equivalent CaCl2 and SrCl2 concentrations. This is the first report of biomineralization of olekminskite and coprecipitation of Sr into vaterite mediated by fungi. These findings suggest that urease-positive fungi could play an important role in the environmental fate, bioremediation or biorecovery of Sr or other metals and radionuclides that form insoluble carbonates.

  6. Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.

    Directory of Open Access Journals (Sweden)

    Dale N Richardson

    Full Text Available Alternative splicing (AS of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes. AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms.

  7. Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots.

    Science.gov (United States)

    Menichelli, Elena; Wu, Joann; Campbell, Zachary T; Wickens, Marvin; Williamson, James R

    2013-02-22

    Caenorhabditis elegans CPB-1 (cytoplasmic polyadenylation element binding protein homolog-1) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms, CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1⋅FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a 10-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective-3). PUF proteins are characterized by the presence of eight α-helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats 7 and 8. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.

  8. 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.

  9. 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.

  10. The physiological expression of scavenger receptor SR-B1 in canine endometrial and placental epithelial cells and its potential involvement in pathogenesis of pyometra.

    Science.gov (United States)

    Gabriel, C; Becher-Deichsel, A; Hlavaty, J; Mair, G; Walter, I

    2016-06-01

    Pyometra, the purulent inflammation of the uterus, is a common uterine disease of bitches that has potentially life-threatening consequences. The opportunistic bacterial infection of the uterus often progresses into the serious systemic inflammatory response syndrome. In a previous study, we characterized epithelial foam cells in the canine endometrial surface occurring in metestrus, and we regularly observed pronounced epithelial foam-cell formations in pyometra-affected uteri. Therefore, it was assumed that the mechanism behind lipid droplet accumulation in surface epithelial cells might even increase bacterial binding capacity and promote pyometra development. Lipid droplet accumulation in epithelial cells is accomplished via specialized lipid receptors called scavenger receptors (SR). Scavenger receptor class B type 1 (SR-B1) is an important receptor for lipid accumulation in diverse cell types, but it is also a strong binding partner for bacteria, and thereby enhances bacterial adhesion and clinical signs of systemic inflammatory response syndrome. In the present study, after the isolation of metestrous surface epithelial cells from canine uteri by laser capture microdissection, SR-B1 was identified at the messenger RNA (mRNA) level by quantitative real time polymerase chain reaction and also at the protein level by means of immunohistochemistry. In pyometra-affected uteri, SR-B1 mRNA expression was higher than that in the healthy control samples, and SR-B1 protein was expressed in the surface and crypt epithelial cells. Furthermore, to understand the physiological role of SR-B1 expression in the metestrus surface epithelial cells, we investigated its expression in the epithelial cells of the glandular chambers of canine placenta in different stages of gestation because these cells are also characterized by lipid droplet accumulation. SR-B1 was present in the placental epithelial cells of the glandular chambers from 25 to 30 and 45 to 50 days of gestation

  11. Genome-wide analysis of small secreted cysteine-rich proteins identifies candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Science.gov (United States)

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight ...

  12. The Aryl-Hydrocarbon Receptor Protein Interaction Network (AHR-PIN as Identified by Tandem Affinity Purification (TAP and Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Dorothy M. Tappenden

    2013-01-01

    Full Text Available The aryl-hydrocarbon receptor (AHR, a ligand activated PAS superfamily transcription factor, mediates most, if not all, of the toxicity induced upon exposure to various dioxins, dibenzofurans, and planar polyhalogenated biphenyls. While AHR-mediated gene regulation plays a central role in the toxic response to dioxin exposure, a comprehensive understanding of AHR biology remains elusive. AHR-mediated signaling starts in the cytoplasm, where the receptor can be found in a complex with the heat shock protein of 90 kDa (Hsp90 and the immunophilin-like protein, aryl-hydrocarbon receptor-interacting protein (AIP. The role these chaperones and other putative interactors of the AHR play in the toxic response is not known. To more comprehensively define the AHR-protein interaction network (AHR-PIN and identify other potential pathways involved in the toxic response, a proteomic approach was undertaken. Using tandem affinity purification (TAP and mass spectrometry we have identified several novel protein interactions with the AHR. These interactions physically link the AHR to proteins involved in the immune and cellular stress responses, gene regulation not mediated directly via the traditional AHR:ARNT heterodimer, and mitochondrial function. This new insight into the AHR signaling network identifies possible secondary signaling pathways involved in xenobiotic-induced toxicity.

  13. Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

    Science.gov (United States)

    Susnea, Iuliana; Bunk, Sebastian; Wendel, Albrecht; Hermann, Corinna; Przybylski, Michael

    2011-04-01

    We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

  14. Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer

    National Research Council Canada - National Science Library

    HATAKEYAMA, Masanori

    2017-01-01

    .... The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs...

  15. PRODUCTION OF PLANTARCIN BY LACTOBACILLUS PLANTARUM SR18

    Directory of Open Access Journals (Sweden)

    Wagih El-Shouny

    2012-06-01

    Full Text Available Out of 86 lactobacilli previously screened in our laboratory, Lactobacillus plantarum SR18 isolated from yoghurt revealed the largest detected inhibition zone against the selected indicator Streptococcus salivarius 5. The obtained electrophoretic patterns revealed that L. plantarum SR18 was free from plasmids. Exposure of 6 h growing L. plantarum culture to T-8M, 3B ultraviolet B lamp (8w, 220v & 312 nm for 2 h and subsequent growth for further 24 h resulted in an increase of cell-bound bacteriocin titer reached 2 fold at 12 h. Whereas bacteriocin secreted in the culture filtrate was not affected by UV irradiation. Plantarcin SR18 production was maximal (12800 AU/ml between 12 and 18 h by incubation of the culture at 37°C and pH 5-7 in candle jar (CO2. The bacteriocin bound to the cells and that secreted into the culture filtrate of L. plantarum SR18 were precipitated by 75% ammomium sulphate, dialysed and further purified by Gel filtration on Sephadex G-100. The specific activities (AU/mg protein were increased by a factor of about 5.3 and 2.35 for plantarcins purified from proteins bound to the cell of L. plantarum SR18 (plantarcin SR18 a and that secreted into the culture filtrate (plantarcin SR18 b, respectively. Gel filtration of plantarcin SR18a resulted in moderate antibacterial activity (3200 AU/ml and very high activity (25600 AU/ml of plantarcin SR18b.

  16. A method to identify protein antigens of Dermanyssus gallinae for the protection of birds from poultry mites.

    Science.gov (United States)

    Makert, Gustavo R; Vorbrüggen, Susanne; Krautwald-Junghanns, Maria-Elisabeth; Voss, Matthias; Sohn, Kai; Buschmann, Tilo; Ulbert, Sebastian

    2016-07-01

    The poultry red mite (PRM) Dermanyssus gallinae causes high economic losses and is among the most important parasites in poultry farming worldwide. Different chemical, physical, and biological strategies try to control the expansion of PRM. However, effective solutions to this problem still have to be found. Here, we present a method for the development of an immunological control strategy, based on the identification of mite protein antigens which elicit antibodies with anti-mite activity in the immunized chicken. Hens were immunized with different PRM protein extracts formulated with two different adjuvants, and IgY-antibodies were isolated from the eggs. A PRM in vitro feeding assay which used chicken blood spiked with these IgY-preparations was used to detect antibodies which caused PRM mortality. In vitro feeding of mites with IgY isolated from hens immunized with PRM extract formulated with one of the adjuvants showed a statistically significant increase in the mortality as compared to control mites. After the separation of total PRM extracts in two-dimensional gels, several protein spots were recognized by such IgY preparations. Ten protein spots were subjected to mass spectrometry (MS/MS) for the identification of the corresponding proteins. Complete protein sequences were deduced from genomic and transcriptomic assemblies derived from high throughput sequencing of total PRM DNA and RNA. The results may contribute to the development of an immunological control strategy of D. gallinae.

  17. PCR and RFLP analyses based on the ribosomal protein operon

    Science.gov (United States)

    Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16Sr RNA gene. RFLP analysis of 16Sr RNA gene sequences has identified 31 16Sr RNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can however, become more refin...

  18. Transportin-SR is required for proper splicing of resistance genes and plant immunity.

    Directory of Open Access Journals (Sweden)

    Shaohua Xu

    2011-06-01

    Full Text Available Transportin-SR (TRN-SR is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14, a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R protein snc1 (suppressor of npr1-1, constitutive 1. MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.

  19. Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels

    OpenAIRE

    Portelli, Michael; Siedlinski, Mateusz; Stewart, Ceri E.; Postma, Dirkje S; Nieuwenhuis, Maartje A.; Vonk, Judith M; Nurnberg, Peter; Altmuller, Janine; Moffatt, M. F.; Wardlaw, Andrew J.; Parker, Stuart G; Connolly, Martin J.; Koppelman, Gerard H; Sayers, Ian

    2014-01-01

    The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonar...

  20. 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.

  1. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

    Directory of Open Access Journals (Sweden)

    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  2. 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.

  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. In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

    Science.gov (United States)

    Zheng, Aihua; Umashankar, Mahadevaiah; Kielian, Margaret

    2010-01-01

    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. PMID:20975939

  5. GPCR-GIA: a web-server for identifying G-protein coupled receptors and their families with grey incidence analysis.

    Science.gov (United States)

    Lin, Wei-Zhong; Xiao, Xuan; Chou, Kuo-Chen

    2009-11-01

    G-protein-coupled receptors (GPCRs) play fundamental roles in regulating various physiological processes as well as the activity of virtually all cells. Different GPCR families are responsible for different functions. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop an automated method to address the two problems: given the sequence of a query protein, can we identify whether it is a GPCR? If it is, what family class does it belong to? Here, a two-layer ensemble classifier called GPCR-GIA was proposed by introducing a novel scale called 'grey incident degree'. The overall success rate by GPCR-GIA in identifying GPCR and non-GPCR was about 95%, and that in identifying the GPCRs among their nine family classes was about 80%. These rates were obtained by the jackknife cross-validation tests on the stringent benchmark data sets where none of the proteins has > or = 50% pairwise sequence identity to any other in a same class. Moreover, a user-friendly web-server was established at http://218.65.61.89:8080/bioinfo/GPCR-GIA. For user's convenience, a step-by-step guide on how to use the GPCR-GIA web server is provided. Generally speaking, one can get the desired two-level results in around 10 s for a query protein sequence of 300-400 amino acids; the longer the sequence is, the more time that is needed.

  6. Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

    Science.gov (United States)

    Kusch, Kathrin; Uecker, Marina; Liepold, Thomas; Möbius, Wiebke; Hoffmann, Christian; Neumann, Heinz; Werner, Hauke B.; Jahn, Olaf

    2017-01-01

    Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions. PMID:28248254

  7. Alanine scanning of cucumber mosaic virus (CMV 2b protein identifies different positions for cell-to-cell movement and gene silencing suppressor activity.

    Directory of Open Access Journals (Sweden)

    Katalin Nemes

    Full Text Available The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA, and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA, which are not essential for suppressor activity.

  8. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor.

    Science.gov (United States)

    Martin, Laetitia B B; Sherwood, Robert W; Nicklay, Joshua J; Yang, Yong; Muratore-Schroeder, Tara L; Anderson, Elizabeth T; Thannhauser, Theodore W; Rose, Jocelyn K C; Zhang, Sheng

    2016-08-01

    We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral library consisting of 11 753 unique peptides, corresponding to 2338 tomato protein groups, was used and the DIA analysis was performed at the MS1 level utilizing narrow mass windows for extraction with Skyline 2.6 software. We identified a total of 1140 proteins, 67 of which had expression levels that differed significantly between the cd2 tomato mutant and the wild-type cultivar M82. Differentially expressed proteins including a key protein involved in cutin biosynthesis, were selected for validation by target SRM/MRM and by Western blot analysis. In addition to confirming a role for CD2 in regulating cuticle formation, the results also revealed that CD2 influences pathways associated with cell wall biology, anthocyanin biosynthesis, plant development, and responses to stress, which complements findings of earlier RNA-Seq experiments. Our results provide new insights into molecular processes and aspects of fruit biology associated with CD2 function, and demonstrate that the WiSIM-DIA is an effective quantitative approach for global protein identifications.

  9. Meta-analysis of genome-wide association studies in >80 000 subjects identifies multiple loci for C-reactive protein levels

    NARCIS (Netherlands)

    Dehghan, Abbas; Dupuis, Josée; Barbalic, Maja; Bis, Joshua C; Eiriksdottir, Gudny; Lu, Chen; Pellikka, Niina; Wallaschofski, Henri; Kettunen, Johannes; Henneman, Peter; Baumert, Jens; Strachan, David P; Fuchsberger, Christian; Vitart, Veronique; Wilson, James F; Paré, Guillaume; Naitza, Silvia; Rudock, Megan E; Surakka, Ida; de Geus, Eco J C; Alizadeh, Behrooz Z; Guralnik, Jack; Shuldiner, Alan; Tanaka, Toshiko; Zee, Robert Y L; Schnabel, Renate B; Nambi, Vijay; Kavousi, Maryam; Ripatti, Samuli; Nauck, Matthias; Smith, Nicholas L; Smith, Albert V; Sundvall, Jouko; Scheet, Paul; Liu, Yongmei; Ruokonen, Aimo; Rose, Lynda M; Larson, Martin G; Hoogeveen, Ron C; Freimer, Nelson B; Teumer, Alexander; Tracy, Russell P; Launer, Lenore J; Buring, Julie E; Yamamoto, Jennifer F; Folsom, Aaron R; Sijbrands, Eric J G; Pankow, James; Elliott, Paul; Keaney, John F; Sun, Wei; Sarin, Antti-Pekka; Fontes, João D; Badola, Sunita; Astor, Brad C; Hofman, Albert; Pouta, Anneli; Werdan, Karl; Greiser, Karin H; Kuss, Oliver; Meyer zu Schwabedissen, Henriette E; Thiery, Joachim; Jamshidi, Yalda; Nolte, Ilja M; Soranzo, Nicole; Spector, Timothy D; Völzke, Henry; Parker, Alexander N; Aspelund, Thor; Bates, David; Young, Lauren; Tsui, Kim; Siscovick, David S; Guo, Xiuqing; Rotter, Jerome I; Uda, Manuela; Schlessinger, David; Rudan, Igor; Hicks, Andrew A; Penninx, Brenda W; Thorand, Barbara; Gieger, Christian; Coresh, Joe; Willemsen, Gonneke; Harris, Tamara B; Uitterlinden, Andre G; Järvelin, Marjo-Riitta; Rice, Kenneth; Radke, Dörte; Salomaa, Veikko; Willems van Dijk, Ko; Boerwinkle, Eric; Vasan, Ramachandran S; Ferrucci, Luigi; Gibson, Quince D; Bandinelli, Stefania; Snieder, Harold; Boomsma, Dorret I; Xiao, Xiangjun; Campbell, Harry; Hayward, Caroline; Pramstaller, Peter P; van Duijn, Cornelia M; Peltonen, Leena; Psaty, Bruce M; Gudnason, Vilmundur; Ridker, Paul M; Homuth, Georg; Koenig, Wolfgang; Ballantyne, Christie M; Witteman, Jacqueline C M; Benjamin, Emelia J; Perola, Markus; Chasman, Daniel I

    2011-01-01

    BACKGROUND: C-reactive protein (CRP) is a heritable marker of chronic inflammation that is strongly associated with cardiovascular disease. We sought to identify genetic variants that are associated with CRP levels. METHODS AND RESULTS: We performed a genome-wide association analysis of CRP in 66 18

  10. Meta-Analysis of Genome-Wide Association Studies in > 80 000 Subjects Identifies Multiple Loci for C-Reactive Protein Levels

    NARCIS (Netherlands)

    Dehghan, Abbas; Dupuis, Josee; Barbalic, Maja; Bis, Joshua C.; Eiriksdottir, Gudny; Lu, Chen; Pellikka, Niina; Wallaschofski, Henri; Kettunen, Johannes; Henneman, Peter; Baumert, Jens; Strachan, David P.; Fuchsberger, Christian; Vitart, Veronique; Wilson, James F.; Pare, Guillaume; Naitza, Silvia; Rudock, Megan E.; Surakka, Ida; de Geus, Eco J. C.; Alizadeh, Behrooz Z.; Guralnik, Jack; Shuldiner, Alan; Tanaka, Toshiko; Zee, Robert Y. L.; Schnabel, Renate B.; Nambi, Vijay; Kavousi, Maryam; Ripatti, Samuli; Nauck, Matthias; Smith, Nicholas L.; Smith, Albert V.; Sundvall, Jouko; Scheet, Paul; Liu, Yongmei; Ruokonen, Aimo; Rose, Lynda M.; Larson, Martin G.; Hoogeveen, Ron C.; Freimer, Nelson B.; Teumer, Alexander; Tracy, Russell P.; Launer, Lenore J.; Buring, Julie E.; Yamamoto, Jennifer F.; Folsom, Aaron R.; Sijbrands, Eric J. G.; Pankow, James; Elliott, Paul; Keaney, John F.; Sun, Wei; Sarin, Antti-Pekka; Fontes, Joao D.; Badola, Sunita; Astor, Brad C.; Hofman, Albert; Pouta, Anneli; Werdan, Karl; Greiser, Karin H.; Kuss, Oliver; Schwabedissen, Henriette E. Meyer Zu; Thiery, Joachim; Jamshidi, Yalda; Nolte, Ilja M.; Soranzo, Nicole; Spector, Timothy D.; Voelzke, Henry; Parker, Alexander N.; Aspelund, Thor; Bates, David; Young, Lauren; Tsui, Kim; Siscovick, David S.; Guo, Xiuqing; Rotter, Jerome I.; Uda, Manuela; Schlessinger, David; Rudan, Igor; Hicks, Andrew A.; Penninx, Brenda W.; Thorand, Barbara; Gieger, Christian; Coresh, Joe; Willemsen, Gonneke; Harris, Tamara B.; Uitterlinden, Andre G.; Jaervelin, Marjo-Riitta; Rice, Kenneth; Radke, Doerte; Salomaa, Veikko; van Dijk, Ko Willems; Boerwinkle, Eric; Vasan, Ramachandran S.; Ferrucci, Luigi; Gibson, Quince D.; Bandinelli, Stefania; Snieder, Harold; Boomsma, Dorret I.; Xiao, Xiangjun; Campbell, Harry; Hayward, Caroline; Pramstaller, Peter P.; van Duijn, Cornelia M.; Peltonen, Leena; Psaty, Bruce M.; Gudnason, Vilmundur; Ridker, Paul M.; Homuth, Georg; Koenig, Wolfgang; Ballantyne, Christie M.; Witteman, Jacqueline C. M.; Benjamin, Emelia J.; Perola, Markus; Chasman, Daniel I.

    2011-01-01

    Background-C-reactive protein (CRP) is a heritable marker of chronic inflammation that is strongly associated with cardiovascular disease. We sought to identify genetic variants that are associated with CRP levels. Methods and Results-We performed a genome-wide association analysis of CRP in 66 185

  11. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor

    Science.gov (United States)

    We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral l...

  12. A 1-year lifestyle intervention for weight loss in individuals with type 2 diabetes reduces high C-reactive protein levels and identifies metabolic predictors of change

    Science.gov (United States)

    OBJECTIVE: We examined whether a 1-year intensive lifestyle intervention (ILI) for weight loss reduced elevated high-sensitivity C-reactive protein (hs-CRP) levels in obese individuals with diabetes and identified metabolic and fitness predictors of hs-CRP change. RESEARCH DESIGN AND METHODS: Look A...

  13. 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.

  14. Meta-analysis of genome-wide association studies in >80 000 subjects identifies multiple loci for C-reactive protein levels

    NARCIS (Netherlands)

    Dehghan, Abbas; Dupuis, Josée; Barbalic, Maja; Bis, Joshua C.; Eiriksdottir, Gudny; Lu, Chen; Pellikka, Niina; Wallaschofski, Henri; Kettunen, Johannes; Henneman, Peter; Baumert, Jens; Strachan, David P.; Fuchsberger, Christian; Vitart, Veronique; Wilson, James F.; Paré, Guillaume; Naitza, Silvia; Rudock, Megan E.; Surakka, Ida; De Geus, Eco J. C.; Alizadeh, Behrooz Z.; Guralnik, Jack; Shuldiner, Alan; Tanaka, Toshiko; Zee, Robert Y. L.; Schnabel, Renate B.; Nambi, Vijay; Kavousi, Maryam; Ripatti, Samuli; Nauck, Matthias; Smith, Nicholas L.; Smith, Albert V.; Sundvall, Jouko; Scheet, Paul; Liu, Yongmei; Ruokonen, Aimo; Rose, Lynda M.; Larson, Martin G.; Hoogeveen, Ron C.; Freimer, Nelson B.; Teumer, Alexander; Tracy, Russell P.; Launer, Lenore J.; Buring, Julie E.; Yamamoto, Jennifer F.; Folsom, Aaron R.; Sijbrands, Eric J. G.; Pankow, James; Elliott, Paul; Keaney, John F.; Sun, Wei; Sarin, Antti-Pekka; Fontes, João D.; Badola, Sunita; Astor, Brad C.; Hofman, Albert; Pouta, Anneli; Werdan, Karl; Greiser, Karin H.; Kuss, Oliver; Meyer Zu Schwabedissen, Henriette E.; Thiery, Joachim; Jamshidi, Yalda; Nolte, Ilja M.; Soranzo, Nicole; Spector, Timothy D.; Völzke, Henry; Parker, Alexander N.; Aspelund, Thor; Bates, David; Young, Lauren; Tsui, Kim; Siscovick, David S.; Guo, Xiuqing; Rotter, Jerome I.; Uda, Manuela; Schlessinger, David; Rudan, Igor; Hicks, Andrew A.; Penninx, Brenda W.; Thorand, Barbara; Gieger, Christian; Coresh, Joe; Willemsen, Gonneke; Harris, Tamara B.; Uitterlinden, Andre G.; Järvelin, Marjo-Riitta; Rice, Kenneth; Radke, Dörte; Salomaa, Veikko; Willems Van Dijk, Ko; Boerwinkle, Eric; Vasan, Ramachandran S.; Ferrucci, Luigi; Gibson, Quince D.; Bandinelli, Stefania; Snieder, Harold; Boomsma, Dorret I.; Xiao, Xiangjun; Campbell, Harry; Hayward, Caroline; Pramstaller, Peter P.; Van Duijn, Cornelia M.; Peltonen, Leena; Psaty, Bruce M.; Gudnason, Vilmundur; Ridker, Paul M.; Homuth, Georg; Koenig, Wolfgang; Ballantyne, Christie M.; Witteman, Jacqueline C. M.; Benjamin, Emelia J.; Perola, Markus; Chasman, Daniel I.

    2011-01-01

    BACKGROUND - C-reactive protein (CRP) is a heritable marker of chronic inflammation that is strongly associated with cardiovascular disease. We sought to identify genetic variants that are associated with CRP levels. METHODS AND RESULTS - We performed a genome-wide association analysis of CRP in 66

  15. Identifying Gel-Separated Proteins Using In-Gel Digestion, Mass Spectrometry, and Database Searching: Consider the Chemistry

    Science.gov (United States)

    Albright, Jessica C.; Dassenko, David J.; Mohamed, Essa A.; Beussman, Douglas J.

    2009-01-01

    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important bioanalytical technique in drug discovery, proteomics, and research at the biology-chemistry interface. This is an especially powerful tool when combined with gel separation of proteins and database mining using the mass spectral data. Currently, few hands-on…

  16. 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

  17. Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

    Science.gov (United States)

    Riley, Sean P; Bykowski, Tomasz; Cooley, Anne E; Burns, Logan H; Babb, Kelly; Brissette, Catherine A; Bowman, Amy; Rotondi, Matthew; Miller, M Clarke; DeMoll, Edward; Lim, Kap; Fried, Michael G; Stevenson, Brian

    2009-04-01

    The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

  18. Identifying Gel-Separated Proteins Using In-Gel Digestion, Mass Spectrometry, and Database Searching: Consider the Chemistry

    Science.gov (United States)

    Albright, Jessica C.; Dassenko, David J.; Mohamed, Essa A.; Beussman, Douglas J.

    2009-01-01

    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important bioanalytical technique in drug discovery, proteomics, and research at the biology-chemistry interface. This is an especially powerful tool when combined with gel separation of proteins and database mining using the mass spectral data. Currently, few hands-on…

  19. Differential Proteome Analysis Identifies TGF-β-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model.

    Directory of Open Access Journals (Sweden)

    Misako Sato

    Full Text Available Transforming growth factor-β (TGF-β has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-β antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-β mediates metastasis-promoting effects is poorly understood. Establishment of TGF-β-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-β oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-β receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-β pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-β tumor-promoting activity.

  20. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.

    Science.gov (United States)

    Alfaro, Joshua F; Gong, Cheng-Xin; Monroe, Matthew E; Aldrich, Joshua T; Clauss, Therese R W; Purvine, Samuel O; Wang, Zihao; Camp, David G; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W; Hunt, Donald F; Yang, Feng; Smith, Richard D

    2012-05-01

    O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

  1. Tandem Mass Spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

    Energy Technology Data Exchange (ETDEWEB)

    Alfaro, Joshua F.; Gong, Cheng-Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese RW; Purvine, Samuel O.; Wang, Zihao; Camp, David G.; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.

    2012-05-08

    O-Linked N-Acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of Ser and Thr residues on cytosolic and nuclear proteins found in all higher eukaryotes. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing {approx}100 {micro}g of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites on Ser and Thr residues in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located in close proximity to known phosphorylation sites. These findings support the proposed regulatory crosstalk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-{beta}-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, thus representing the first evidence for extracellular O-GlcNAcylation in mammalian systems by the ER-resident O-GlcNAc transferase (EOGT). We also report a GlcNAc-{beta}-1,3-Fuc-{alpha}-1-O-Thr modification on the EGF-like repeat of the Versican core protein, a novel substrate of Fringe {beta}1,3-N-acetylglucosaminyltransferases.

  2. 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.

  3. A novel highly divergent protein family identified from a viviparous insect by RNA-seq analysis: a potential target for tsetse fly-specific abortifacients.

    Directory of Open Access Journals (Sweden)

    Joshua B Benoit

    2014-04-01

    Full Text Available In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1 and seven new genes (mgp4-10. The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during

  4. Molecular docking and dynamic studies of human growth factor receptorbound protein (Grb 2 insights to identify novel inhibitors

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    Sandeep S

    2016-10-01

    Full Text Available Background: Human growth factor receptor bound protein-2 (Grb 2 involves in initiation of kinase signaling by Son of Sevenless (SOS and activates mitogen activated protein kinase pathway. Grb2 overexpress during cancerous condition hence it emerged as a potent target for various cancers. Material and Methods: Seven pharmacophores were developed from seven co-crystal structures of Grb2 and applied for common pharmacophore hypothesis. Two common pharmacophore hypothesis (CPH models were screened and hits were applied for docking and free energy [G] calculations. Results: Two leads were proposed from docking and G analysis. Energy of the system, RMSD, RMSF, hydrogen bonds and water bridges of lead1 was better than the co-crystal ligand during 50 ns molecular dynamics simulations. Discussion: Two leads are interacting with Src homology 2 (SH2 domain of Grb2 and blocking the function of Grb2.

  5. 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

    NARCIS (Netherlands)

    Heidema, A.G.; Thissen, U.; Boer, J.M.; Bouwman, F.G.; Feskens, E.J.M.; Mariman, E.C.

    2009-01-01

    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

  6. 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

    2015-12-01

    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

  7. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    Directory of Open Access Journals (Sweden)

    Karyna eRosario

    2015-07-01

    Full Text Available Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA viruses that encode a conserved replication initiator protein (Rep in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs, which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  8. Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

    Science.gov (United States)

    Xiao, Xiang-Hong; Miao, Hui-Min; Xu, Yi-Gang; Zhang, Jing-Yu; Chai, Long-Hui; Xu, Jia-Jia

    2014-04-01

    The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

  9. Substrate profiling of human vaccinia-related kinases identifies coilin, a Cajal body nuclear protein, as a phosphorylation target with neurological implications.

    Science.gov (United States)

    Sanz-García, Marta; Vázquez-Cedeira, Marta; Kellerman, Efrat; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A

    2011-12-21

    Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. In general, knowledge on kinase specificity is restricted to substrates identified in the context of specific cellular responses, but kinases are likely to have multiple additional substrates and be integrated in signaling networks that might be spatially and temporally different, and in which protein complexes and subcellular localization can play an important role. In this report the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-array containing 1080 sequences phosphorylated in known signaling pathways has been studied. The two kinases identify a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues in peptide targets. Linear peptide arrays are therefore a useful approach in the characterization of kinases and substrate identification, which can contribute to delineate the signaling network in which VRK proteins participate. One of these target proteins is coilin; a basic protein located in nuclear Cajal bodies. Coilin is phosphorylated in Ser184 by both VRK1 and VRK2. Coilin colocalizes and interacts with VRK1 in Cajal bodies, but not with the mutant VRK1 (R358X). VRK1 (R358X) is less active than VRK1. Altered regulation of coilin might be implicated in several neurological diseases such as ataxias and spinal muscular atrophies.

  10. A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

    Directory of Open Access Journals (Sweden)

    Nadia Korfali

    Full Text Available Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/- cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

  11. Complement-activated oligodendroglia: a new pathogenic entity identified by immunostaining with antibodies to human complement proteins C3d and C4d.

    Science.gov (United States)

    Yamada, T; Akiyama, H; McGeer, P L

    1990-05-04

    Clusters of oligodendroglial fibers were identified immunohistochemically in human brain tissue with antibodies to the complement proteins C3d and C4d in several neurological disorders. These included Pick's, Huntington's, Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy and Shy-Drager syndrome. These complement-activated oligodendroglia occurred in selected areas of gray and white matter. They were rarely observed in control tissue. Immunogold electron microscopy established that the C4d antibody was attached to degenerating myelin sheaths. These data indicate attachment of classical complement pathway proteins to selective oligodendroglia in several neurological disorders.

  12. Identifying interacting proteins of a Caenorhabditis elegans voltage-gated chloride channel CLH-1 using GFP-Trap and mass spectrometry.

    Science.gov (United States)

    Zhou, Zi-Liang; Jiang, Jing; Yin, Jiang-An; Cai, Shi-Qing

    2014-06-25

    Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride ch