714 research outputs found

    PPDB, the Plant Proteomics Database at Cornell

    Get PDF
    The Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.edu), launched in 2004, provides an integrated resource for experimentally identified proteins in Arabidopsis and maize (Zea mays). Internal BLAST alignments link maize and Arabidopsis information. Experimental identification is based on in-house mass spectrometry (MS) of cell type-specific proteomes (maize), or specific subcellular proteomes (e.g. chloroplasts, thylakoids, nucleoids) and total leaf proteome samples (maize and Arabidopsis). So far more than 5000 accessions both in maize and Arabidopsis have been identified. In addition, more than 80 published Arabidopsis proteome datasets from subcellular compartments or organs are stored in PPDB and linked to each locus. Using MS-derived information and literature, more than 1500 Arabidopsis proteins have a manually assigned subcellular location, with a strong emphasis on plastid proteins. Additional new features of PPDB include searchable posttranslational modifications and searchable experimental proteotypic peptides and spectral count information for each identified accession based on in-house experiments. Various search methods are provided to extract more than 40 data types for each accession and to extract accessions for different functional categories or curated subcellular localizations. Protein report pages for each accession provide comprehensive overviews, including predicted protein properties, with hyperlinks to the most relevant databases

    A Mammalian Mediator Subunit that Shares Properties with Saccharomyces cerevisiae Mediator Subunit Cse2

    Get PDF
    The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193open reading frame. In this study, we present direct biochemical evidence that the FLJ10193protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex

    Abacus: A computational tool for extracting and pre-processing spectral count data for label-free quantitative proteomic analysis

    Full text link
    We describe Abacus, a computational tool for extracting spectral counts from MS/MS data sets. The program aggregates data from multiple experiments, adjusts spectral counts to accurately account for peptides shared across multiple proteins, and performs common normalization steps. It can also output the spectral count data at the gene level, thus simplifying the integration and comparison between gene and protein expression data. Abacus is compatible with the widely used Trans-Proteomic Pipeline suite of tools and comes with a graphical user interface making it easy to interact with the program. The main aim of Abacus is to streamline the analysis of spectral count data by providing an automated, easy to use solution for extracting this information from proteomic data sets for subsequent, more sophisticated statistical analysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83477/1/1340_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/83477/2/pmic_201000650_sm_SupplInfo.pd

    Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen

    Get PDF
    Abstract Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO2 concentrations and CO2 concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO2 assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO2. The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.</jats:p

    Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

    Get PDF
    A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato(Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration)and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome

    A comparative phosphoproteomic analysis of a human tumor metastasis model using a label-free quantitative approach

    Full text link
    Alterations in cellular phosphorylation patterns have been implicated in a number of diseases, including cancer, through multiple mechanisms. Herein we present a survey of the phosphorylation profiles of an isogenic pair of human cancer cell lines with opposite metastatic phenotype. Phosphopeptides were enriched from tumor cell lysates with titanium dioxide and zirconium dioxide, and identified with nano-LC-MS/MS using an automatic cross-validation of MS/MS and MS/MS/MS (MS2+MS3) data-dependent neutral loss method. A spectral counting quantitative strategy was applied to the two cell line samples on the MS2-only scan, which was implemented successively after each MS2+MS3 scan in the same sample. For all regulated phosphopeptides reported by spectral counting analysis, sequence and phosphorylation site assignments were validated by a MS2+MS3 data-dependent neutral loss method. With this approach, we identified over 70 phosphorylated sites on 27 phosphoproteins as being differentially expressed with respect to tumor cell phenotype. The altered expression levels of proteins identified by LC-MS/MS were validated using Western blotting. Using network pathway analysis, we observed that the majority of the differentially expressed proteins were highly interconnected and belong to two major intracellular signaling pathways. Our findings suggest that the phosphorylation of isoform A of lamin A/C and GTPase activating protein binding protein 1 is associated with metastatic propensity. The study demonstrates a quantitative and comparative proteomics strategy to identify differential phosphorylation patterns in complex biological samples.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75766/1/1842_ftp.pd

    The Inhibition of Polo Kinase by Matrimony Maintains G2 Arrest in the Meiotic Cell Cycle

    Get PDF
    Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase

    Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling

    Get PDF
    Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762516/Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.This work was supported by NIAID Clinical Proteomics Center Grant HHSN272200800048C (to A.R.B.), University of Texas Medical Branch Clinical and Translational Science Award (CTSA) UL1TR000071 (to A.R.B.), and NIEHS Grant P30 ES006676 (to A.R.B.).info:eu-repo/semantics/publishedVersio
    corecore