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ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses

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Abstract

Protein mono-ADP-ribosylation is a reversible post-translational modification of cellular proteins. This scheme of amino-acid modification is used not only by bacterial toxins to attack host cells, but also by endogenous ADP-ribosyltransferases (ARTs) in mammalian cells. These latter ARTs include members of three different families of proteins: the well characterised arginine-specific ecto-enzymes (ARTCs), two sirtuins, and some members of the poly(ADP-ribose) polymerase (PARP/ARTD) family. In the present study, we demonstrate that human ARTC1 is localised to the endoplasmic reticulum (ER), in contrast to the previously characterised ARTC proteins, which are typical GPI-anchored ecto-enzymes. Moreover, using the “macro domain” cognitive binding module to identify ADP-ribosylated proteins, we show here that the ER luminal chaperone GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) is a cellular target of human ARTC1 and hamster ARTC2. We further developed a procedure to visualise ADP-ribosylated proteins using immunofluorescence. With this approach, in cells overexpressing ARTC1, we detected staining of the ER that co-localises with GRP78/BiP, thus confirming that this modification occurs in living cells. In line with the key role of GRP78/BiP in the ER stress response system, we provide evidence here that ARTC1 is activated during the ER stress response, which results in acute ADP-ribosylation of GRP78/BiP paralleling translational inhibition. Thus, this identification of ARTC1 as a regulator of GRP78/BiP defines a novel, previously unsuspected, player in GRP78-mediated ER stress responses.

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Abbreviations

ART:

ADP-ribosyltransferase

CHO cells:

Chinese hamster ovary cells

DTT:

Dithiothreitol

ER:

Endoplasmic reticulum

GST:

Glutatione S-transferase

HeLa cells:

Human cervix adenocarcinoma cells

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Acknowledgments

The authors thank Antonio Tamburro for protein identification by MALDI-MS analysis, Elena Fontana for preparation of Figures, Marco Scardapane for his advice on Statistical analysis and Chris Berrie for editorial assistance. We thank David Ron (University of Cambridege, UK) for providing us the Flag-GRP78 plasmid. We gratefully acknowledge the financial support of the Banca Popolare dell’Emilia Romagna, the Italian Association for Cancer Research (AIRC IG 11652), and the Deutsche Forschungsgemeinschaft (SFB877-A5).

Author information

Author notes

  1. Simone Di Paola

    Present address: Telethon Institute of Genetics and Medicine, Via Pietro Castellino 111, 80131, Naples, Italy

  2. Monica Giannotta

    Present address: Unit of Vascular Biology, The FIRC Institute of Molecular Oncology Foundation, Milan, Italy

  3. Carmen Ruggiero

    Present address: Associated International Laboratory (LIA) NEOGENEX CNRS, University of Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560, Valbonne, France

Authors and Affiliations

  1. Laboratory of G-Protein-mediated Signalling, Department of Cellular and Translational Pharmacology, Mario Negri Sud Foundation, Via Nazionale 8/A, 66030, Santa Maria Imbaro, CH, Italy

    Gaia Fabrizio, Simone Di Paola, Annalisa Stilla & Maria Di Girolamo

  2. Genomic Approaches to Membrane Traffic Unit, Department of Cellular and Translational Pharmacology, Mario Negri Sud Foundation, Via Nazionale, 8/A, 66030, Santa Maria Imbaro, CH, Italy

    Monica Giannotta, Carmen Ruggiero & Michele Sallese

  3. Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Martinist 52, 20246, Hamburg, Germany

    Stephan Menzel & Friedrich Koch-Nolte

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  1. Gaia Fabrizio
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Correspondence to Maria Di Girolamo.

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G. Fabrizio and S. Di Paola contributed equally to this manuscript.

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Fabrizio, G., Di Paola, S., Stilla, A. et al. ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses. Cell. Mol. Life Sci. 72, 1209–1225 (2015). https://doi.org/10.1007/s00018-014-1745-6

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