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

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.

ARTD10/PARP10 Induces ADP-Ribosylation of GAPDH and Recruits GAPDH into Cytosolic Membrane-Free Cell Bodies When Overexpressed in Mammalian Cells

“…The samples were either analysed by silver staining, and the immunoprecipitated bands were identified by MALDI-TOF mass spectrometry (immunoprecipitation; Figure 1C) or resolved by Western blotting using antibodies raised against GAPDH or the HA tag. The GST pull-down was performed as previously reported [13,40]. Briefly, two mg of total lysates for each sample were solubilised with 1.5 mL RIPA buffer and incubated with a glutathione-Sepharose resin for 2 h at 4 • C, for the preclearing step.…”

“…Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS), fixed for 10 min at room temperature with 4% paraformaldehyde, permeabilised in blocking solution (PBS, 50 mM NH 4 Cl, 0.5% BSA, 0.1% saponin) for 20 min at room temperature, and stained with an anti-HA antibody (Cell Signalling Technology, Leiden, The Netherlands; 1:600 in blocking solution) and an anti-GAPDH antibody (Santa Cruz Biotechnology, Dallas, TX, USA; 1:50 in blocking solution). The GST-mAf1521 immunofluorescence staining was performed as described [13]. Briefly, the cells were fixed, permeabilised and then incubated for 1 h at room temperature with 6 ng of GST-mAf1521.…”

Molecular signal networks and regulating mechanisms of the unfolded protein response

“…Finally, BAP/Sil1 dissociates the BiP/three sensors complex by directly interacting with GRP78/BiP (Chung et al, 2002;Chen et al, 2014a). At the same time, the activity of GRP78/BiP is also regulated by mono-ADP-ribosylation by arginine-specific ectoenzymes (ARTCs) on two arginine residues (R470 and R492) in UPR, which interferes with BiP to bind with the three sensors and augment the UPR signals (Fabrizio et al, 2014). The expressions of all of these interacting proteins are induced through UPR signaling pathways to regulate cooperatively and balance protein processing with the demands of protein synthesis.…”

“…Expressions of caspase 2 and subsequent Bid proteolytic cleavage increase, contributing to apoptotic cell death (Hassler et al, 2012). Furthermore, IRE1-mediated ASK1/JNK activation leads to the cleavage and activation of procaspase 12 (in mouse) or procaspase 4 (in humans) to promote cell death (Fabrizio et al, 2014;.…”

“…(5) IRE1β-mediated ASK1/JNK activation leads to the cleavage and activation of procaspase 12 (in mouse) or procaspase 4 (in humans) to promote cell death (Fabrizio et al, 2014;; furthermore, the IRE1α-TRAF2-ASK1 complex inhibits the Ca 2+ -binding inositol 1,4,5-triphosphate receptor (InsP3R) from binding its inhibitor CIB1, thus inhibiting ER-to-cytosolic efflux of Ca 2+ . The severe UPR induces the dissociation of ASK1 from the IRE1α-TRAF2-ASK1 complex and increases the assembly of ASK1/calcium and integrin binding 1 (CIB1) complex.…”