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

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.

Mono-ADP-ribosylation of the G protein betagamma dimer is modulated by hormones and inhibited by Arf6.

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the ß subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the ßγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gß subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gß subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gß subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gß subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gß subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gß subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gß subunit mono-ADP-ribosylation.

Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome.

Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification.

Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein.

The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in approximately 170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2-mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

Physiological relevance of the endogenous mono(ADP-ribosyl)ation of cellular proteins.

The mono(ADP-ribosyl)ation reaction is a post-translational modification that is catalysed by both bacterial toxins and eukaryotic enzymes, and that results in the transfer of ADP-ribose from betaNAD+ to various acceptor proteins. In mammals, both intracellular and extracellular reactions have been described; the latter are due to glycosylphosphatidylinositol-anchored or secreted enzymes that are able to modify their targets, which include the purinergic receptor P2X7, the defensins and the integrins. Intracellular mono(ADP-ribosyl)ation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78/BiP, the beta-subunit of heterotrimeric G-proteins and glutamate dehydrogenase. The molecular identification of the intracellular enzymes, however, is still missing. A better molecular understanding of this reaction will help in the full definition of its role in cell physiology and pathology.

Identification and partial characterization of Rhizopus nigricans Gß proteins and their expression in the presence of progesterone.

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gß subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gß subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gß from other filamentous fungi, but not from yeast. The presence of more than one Gß subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gß subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gß subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.

Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells.

The mammalian mono-ADP-ribosyltransferases are a family of enzymes related to bacterial toxins that can catalyse both intracellular and extracellular mono-ADP-ribosylation of target proteins involved in different cellular processes, such as cell migration, signalling and inflammation. Here, we report the molecular cloning and functional characterisation of a novel glycosylphosphatidylinositol (GPI)-anchored mono-ADP-ribosyltransferase isoform from Chinese hamster ovary (CHO) cells (cARTC2.1) that has both NAD-glycohydrolase and arginine-specific ADP-ribosyltransferase activities. cARTC2.1 has the R-S-EXE active-site motif that is typical of arginine-specific ADP-ribosyltransferases, with Glu209 as the predicted catalytic amino acid. When over-expressed in CHO cells, the E209G single point mutant of cARTC2.1 cannot hydrolyse NAD(+), although it retains low arginine-specific ADP-ribosyltransferase activity. This ADP-ribosyltransferase activity was abolished only with an additional mutation in the R-S-EXE active-site motif, with both of the glutamate residues of the EKE sequence of cARTC2.1 mutated to glycine (E207/209G). These glutamate-mutated proteins localise to the plasma membrane, as does wild-type cARTC2.1. Thus, the partial or total loss of enzymatic activity of cARTC2.1 that arises from these mutations does not affect its cellular localisation. Importantly, an endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells, while a similar activity cannot be detected in ovarian cancer cells. With respect to this endogenous ecto-ART activity, we have identified two cell populations: ART-positive and ART-negative CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population.