223 research outputs found

    RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response

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    In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3′ poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress. DOI: http://dx.doi.org/10.7554/eLife.09656.00

    ProteomeScout: A repository and analysis resource for post-translational modifications and proteins

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    ProteomeScout (https://proteomescout.wustl.edu) is a resource for the study of proteins and their post-translational modifications (PTMs) consisting of a database of PTMs, a repository for experimental data, an analysis suite for PTM experiments, and a tool for visualizing the relationships between complex protein annotations. The PTM database is a compendium of public PTM data, coupled with user-uploaded experimental data. ProteomeScout provides analysis tools for experimental datasets, including summary views and subset selection, which can identify relationships within subsets of data by testing for statistically significant enrichment of protein annotations. Protein annotations are incorporated in the ProteomeScout database from external resources and include terms such as Gene Ontology annotations, domains, secondary structure and non-synonymous polymorphisms. These annotations are available in the database download, in the analysis tools and in the protein viewer. The protein viewer allows for the simultaneous visualization of annotations in an interactive web graphic, which can be exported in Scalable Vector Graphics (SVG) format. Finally, quantitative data measurements associated with public experiments are also easily viewable within protein records, allowing researchers to see how PTMs change across different contexts. ProteomeScout should prove useful for protein researchers and should benefit the proteomics community by providing a stable repository for PTM experiments

    Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase

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    Background Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. Discussion These findings provide novel insights into the evolution, structure, and function of this unique CA form

    The BioGRID interaction database: 2015 update

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    The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org) is an open access database that houses genetic and protein interactions curated from the primary biomedical literature for all major model organism species and humans. As of September 2014, the BioGRID contains 749 912 interactions as drawn from 43 149 publications that represent 30 model organisms. This interaction count represents a 50% increase compared to our previous 2013 BioGRID update. BioGRID data are freely distributed through partner model organism databases and meta-databases and are directly downloadable in a variety of formats. In addition to general curation of the published literature for the major model species, BioGRID undertakes themed curation projects in areas of particular relevance for biomedical sciences, such as the ubiquitin-proteasome system and various human disease-associated interaction networks. BioGRID curation is coordinated through an Interaction Management System (IMS) that facilitates the compilation interaction records through structured evidence codes, phenotype ontologies, and gene annotation. The BioGRID architecture has been improved in order to support a broader range of interaction and post-translational modification types, to allow the representation of more complex multi-gene/protein interactions, to account for cellular phenotypes through structured ontologies, to expedite curation through semi-automated text-mining approaches, and to enhance curation quality control
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