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Static Clathrin Assemblies at the Peripheral Vacuole—Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia

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      Abstract

      Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain ( GlCHC), dynamin-related protein ( GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.

      Author Summary

      In canonical clathrin mediated endocytosis (CME) models, the concerted action of ca. 50 proteins mediates the uptake of extracellular components. The key player in this process is clathrin which coats transport intermediates called clathrin coated vesicles (CCV). The intestinal parasite Giardia lamblia has undergone extensive remodeling during colonization of the mammalian duodenum. Here, we report on unique features of this parasite’s endocytic system, consisting of fixed peripheral vacuoles (PV) in close proximity to the exposed plasma membrane (PM), with no discernible CCVs. Using state-of-the-art imaging strategies, we show that the surface of Giardia trophozoites is pock-marked with PM invaginations reaching to the underlying PV membrane. Co-immunoprecipitation and analysis of protein dynamics reveal that, in line with the absence of CCVs, giardial clathrin assemblies have no dynamic behavior. CHC still remains associated to AP2 and dynamin, both conserved dynamic CME components, and to a newly identified putative clathrin light chain. The emerging model calls for giardial clathrin organized into static cores surrounded by dynamic interaction partners, and most likely involved in the regulation of fusion between the PM and the PVs in a “kiss-and-flush”-like mechanism. This suggests that Giardia harbors a conceptually novel function for clathrin in endocytosis, which might be a consequence of host-parasite co-evolution.

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      I-TASSER: a unified platform for automated protein structure and function prediction.

      The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission numbers and Gene Ontology terms. An estimate of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.
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        I-TASSER server for protein 3D structure prediction

         Yang Zhang (2008)
        Background Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions. Results An on-line version of I-TASSER is developed at the KU Center for Bioinformatics which has generated protein structure predictions for thousands of modeling requests from more than 35 countries. A scoring function (C-score) based on the relative clustering structural density and the consensus significance score of multiple threading templates is introduced to estimate the accuracy of the I-TASSER predictions. A large-scale benchmark test demonstrates a strong correlation between the C-score and the TM-score (a structural similarity measurement with values in [0, 1]) of the first models with a correlation coefficient of 0.91. Using a C-score cutoff > -1.5 for the models of correct topology, both false positive and false negative rates are below 0.1. Combining C-score and protein length, the accuracy of the I-TASSER models can be predicted with an average error of 0.08 for TM-score and 2 Å for RMSD. Conclusion The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I-TASSER models. The output of the I-TASSER server for each query includes up to five full-length models, the confidence score, the estimated TM-score and RMSD, and the standard deviation of the estimations. The I-TASSER server is freely available to the academic community at .
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          2016 update of the PRIDE database and its related tools

          The PRoteomics IDEntifications (PRIDE) database is one of the world-leading data repositories of mass spectrometry (MS)-based proteomics data. Since the beginning of 2014, PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) is the new PRIDE archival system, replacing the original PRIDE database. Here we summarize the developments in PRIDE resources and related tools since the previous update manuscript in the Database Issue in 2013. PRIDE Archive constitutes a complete redevelopment of the original PRIDE, comprising a new storage backend, data submission system and web interface, among other components. PRIDE Archive supports the most-widely used PSI (Proteomics Standards Initiative) data standard formats (mzML and mzIdentML) and implements the data requirements and guidelines of the ProteomeXchange Consortium. The wide adoption of ProteomeXchange within the community has triggered an unprecedented increase in the number of submitted data sets (around 150 data sets per month). We outline some statistics on the current PRIDE Archive data contents. We also report on the status of the PRIDE related stand-alone tools: PRIDE Inspector, PRIDE Converter 2 and the ProteomeXchange submission tool. Finally, we will give a brief update on the resources under development ‘PRIDE Cluster’ and ‘PRIDE Proteomes’, which provide a complementary view and quality-scored information of the peptide and protein identification data available in PRIDE Archive.
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            Author and article information

            Affiliations
            [1 ]Institute of Parasitology, University of Zurich, Zurich, Switzerland
            [2 ]Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland
            University of California Los Angeles, UNITED STATES
            Author notes

            The authors have declared that no competing interests exist.

            Conceived and designed the experiments: JPZ LC SR AK CF ABH. Performed the experiments: JPZ LC SR AK. Analyzed the data: JPZ LC CF ABH. Contributed reagents/materials/analysis tools: AK. Wrote the paper: JPZ CF ABH.

            [¤]

            Current address: Department of Chemistry and Biochemistry, University of Berne, Berne, Switzerland

            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, CA USA )
            1553-7366
            1553-7374
            20 July 2016
            July 2016
            : 12
            : 7
            27438602 4954726 10.1371/journal.ppat.1005756 PPATHOGENS-D-16-00456
            © 2016 Zumthor et al

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Figures: 11, Tables: 0, Pages: 33
            Product
            Funding
            Funded by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (CH)
            Award ID: 31-140803/1
            Award Recipient :
            Funded by: Forschungskredit der Universität Zürich
            Award ID: 55080507
            Award Recipient :
            Funded by: Forschungskredit der Universität Zürich
            Award ID: K-52201-05-01
            Award Recipient :
            Funded by: Forschungskredit der Universität Zürich
            Award ID: K-52201-06-01
            Award Recipient :
            This work was partially supported by grant nr. 31-140803/1, awarded to ABH by the Swiss National Science Fund ( www.snf.ch). JPZ received grant nrs. 55080507 followed by K-52201-05-01, and CF received grant nr. K-52201-06-01, all as fellowships from the Forschungskredit der Universität Zürich ( www.researchers.uzh.ch). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology and Life Sciences
            Cell Biology
            Cellular Structures and Organelles
            Cell Membranes
            Biology and Life Sciences
            Parasitology
            Parasite Groups
            Apicomplexa
            Trophozoites
            Research and Analysis Methods
            Microscopy
            Light Microscopy
            Fluorescence Recovery after Photobleaching
            Biology and Life Sciences
            Organisms
            Protozoans
            Parasitic Protozoans
            Giardia
            Biology and Life Sciences
            Cell Biology
            Cellular Structures and Organelles
            Biology and Life Sciences
            Cell Biology
            Cellular Structures and Organelles
            Cell Membranes
            Membrane Proteins
            Biology and Life Sciences
            Organisms
            Protozoans
            Parasitic Protozoans
            Giardia
            Giardia Lamblia
            Research and Analysis Methods
            Microscopy
            Electron Microscopy
            Transmission Electron Microscopy
            Custom metadata
            Data are freely retrievable using project accession number PXD003718 and project DOI 10.6019/PXD003718.

            Infectious disease & Microbiology

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