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  1. Article ; Online: Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

    Eva Pauwels / Becky Provinciael / Anita Camps / Enno Hartmann / Kurt Vermeire

    International Journal of Molecular Sciences, Vol 23, Iss 584, p

    2022  Volume 584

    Abstract: One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered ... ...

    Abstract One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.
    Keywords co-translational translocation ; endoplasmic reticulum ; cyclotriazadisulfonamide ; ER quality control ; DNAJC3 ; signal peptide ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 570
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Intracellular flow cytometry complements RT-qPCR detection of circulating SARS-CoV-2 variants of concern

    Emiel Vanhulle / Becky Provinciael / Joren Stroobants / Anita Camps / Piet Maes / Kurt Vermeire

    BioTechniques, Vol 72, Iss 6, Pp 245-

    2022  Volume 254

    Abstract: Basic and antiviral research on SARS-CoV-2 rely on cellular assays of virus replication in vitro. In addition, accurate detection of virus-infected cells and released virus particles is needed to study virus replication and to profile new candidate ... ...

    Abstract Basic and antiviral research on SARS-CoV-2 rely on cellular assays of virus replication in vitro. In addition, accurate detection of virus-infected cells and released virus particles is needed to study virus replication and to profile new candidate antiviral drugs. Here, by flow cytometry, we detect SARS-CoV-2 infection at single cell level and distinguish infected Vero E6 cells from uninfected bystander cells. Furthermore, based on the viral nucleocapsid expression, subpopulations of infected cells that are in an early or late phase of viral replication can be differentiated. Importantly, this flow cytometric technique complements our duplex RT-qPCR detection of viral E and N, and it can be applied to all current SARS-CoV-2 variants of concern, including the highly mutated Omicron variant.
    Keywords antiviral drug ; duplex RT-qPCR ; flow cytometry ; nucleocapsid ; omicron variant ; SARS-CoV-2 ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2022-06-01T00:00:00Z
    Publisher Future Science Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article: Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

    Vermeire, Kurt / Becky Provinciael / Enno Hartmann / Kai-Uwe Kalies / Susanne Allan

    Analytical biochemistry. 2015 Sept. 01, v. 484

    2015  

    Abstract: Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ... ...

    Abstract Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes.
    Keywords cows ; dogs ; glycosylation ; microsomes ; protein transport ; ribonucleases ; sheep ; swine ; vascular cell adhesion molecules
    Language English
    Dates of publication 2015-0901
    Size p. 102-104.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2015.05.019
    Database NAL-Catalogue (AGRICOLA)

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  4. Article ; Online: Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway.

    Wolfgang Klein / Claudia Rutz / Jamina Eckhard / Becky Provinciael / Edgar Specker / Martin Neuenschwander / Gunnar Kleinau / Patrick Scheerer / Jens-Peter von Kries / Marc Nazaré / Kurt Vermeire / Ralf Schülein

    PLoS ONE, Vol 13, Iss 12, p e

    2018  Volume 0208641

    Abstract: The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are ... ...

    Abstract The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2018-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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