Protein ADP-ribosylation is a reversible post-translational modification of cellular proteins that is catalysed by enzymes that transfer one (mono) or several (poly) units of ADP-ribose from β-NAD + to a specific amino acid of the target protein. The most studied member of the ADP-ribosyltransferase family is PARP1 (also known as ADP-ribosyltransferase diphtheria toxin-like 1, ARTD1), which is directly activated by DNA strand breaks and is involved in DNA damage repair, chromatin remodelling and transcriptional regulation. Much less is known about the further 16 members of this family. Among these, ARTD10/PARP10 has been previously characterised as a mono-ADP-ribosyltransferase with a role in cell proliferation and in NF-kB signalling. In the present study, we identified the glycolytic enzyme GAPDH as an interactor and a novel cellular target for ARTD10/PARP10. Moreover, we detected the co-localisation of GAPDH and ARTD10/PARP10 in well-defined cytosolic bodies, which we show here to be membrane-free, rounded structures using immunogold labelling and electron microscopy. Using the cognitive binding module macro domain to visualise ADP-ribosylated proteins by immunofluorescence microscopy in cells over-expressing the ARTD10/PARP10 enzyme, we show that the staining of the ARTD10/PARP10-dependent cytosolic bodies was lost when the cells were treated with compounds that inhibit ARTD10/PARP10, either by directly inhibiting the enzyme or by reducing the cellular NAD + levels. In parallel, the same treatment affected the co-localisation of GAPDH and ARTD10/PARP10, as GAPDH disappeared from the cytosolic cell bodies, which indicates that its presence there depends on the catalytic activity of ARTD10/PARP10. In line with this, in cells over-expressing the ARTD10/PARP10 catalytic domain alone, which we show here to form stress granules, GAPDH was recruited into stress granules. These data identify ARTD10/PARP10 as the enzyme that modifies and recruits GAPDH into cytosolic structures.

Ketoprofen L-lysine salt (KLS), is widely used due to its analgesic efficacy and tolerability, and L-lysine was reported to increase the solubility and the gastric tolerance of ketoprofen. In a recent report, L-lysine salification has been shown to exert a gastroprotective effect due to its specific ability to counteract the NSAIDs-induced oxidative stress and up-regulate gastroprotective proteins. In order to derive further insights into the safety and efficacy profile of KLS, in this study we additionally compared the effect of lysine and arginine, another amino acid counterion commonly used for NSAIDs salification, in control and in ethanol challenged human gastric mucosa model. KLS is widely used for the control of post-surgical pain and for the management of pain and fever in inflammatory conditions in children and adults. It is generally well tolerated in pediatric patients, and data from three studies in >900 children indicate that oral administration is well tolerated when administered for up to 3 weeks after surgery. Since only few studies have so far investigated the effect of ketoprofen on gastric mucosa maintenance and adaptive mechanisms, in the second part of the study we applied the cMap approach to compare ketoprofen-induced and ibuprofen-induced gene expression profiles in order to explore compound-specific targeted biological pathways. Among the several genes exclusively modulated by ketoprofen, our attention was particularly focused on genes involved in the maintenance of gastric mucosa barrier integrity (cell junctions, morphology, and viability). The hypothesis was further validated by Real-time PCR.

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the ␤ subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the ␤␥ 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 G␤ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces G␤ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits G␤ 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 G␤ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of G␤ 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 G␤ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of G␤ subunit mono-ADP-ribosylation.Mono-ADP-ribosylation is a post-translational modification that is catalyzed by bacterial toxins and eukaryotic enzymes and consists of the transfer of the ADP-ribose moiety from ␤-NAD ϩ to a specific amino acid of various cellular proteins (1-3). We have previously demonstrated the occurrence of functional mono-ADP-ribosylation of the heterotrimeric G protein ␤ subunit (4, 5).The heterotrimeric G proteins are the most common transducers for G protein-coupled receptors (GPCRs), 4 the largest family of cell-surface receptors. They transduce biological signals from the cell surface to the intracellular compartment (6, 7). In the classical model of G protein signaling, when GPCRs are activated by ligand binding they catalyze the exchange of the tightly bound GDP for GTP on the ␣ subunit of the heterotrimeric G protein; the binding of GTP activates the G protein and the G␣ subunit dissociates from the G␤␥ dimer. Both the G␣ subunit and the G␤␥ dimer regulate effector proteins, including adenylyl cyclases, phospholipases, MAPK, and ion channels (see Refs. 8, 9 and references therein). Thus, although the G␤␥ dimers were once thought to only be negative regulators of G␣-dependent signaling, they are now well recognized as mediators of receptor signaling in their own right (8,9).We have previously demonstrated that a membrane-associated, arginine-specific mono-ADP-ribosyltransferase modifies residue 129 of the G␤ subunit when it is in its active heterodimeric conformation (4,5,10). This ...

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