G Protein mediated Signalling Laboratory

Santa Maria Imbaro, Italy

G Protein mediated Signalling Laboratory

Santa Maria Imbaro, Italy
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Di Girolamo M.,G Protein mediated Signalling Laboratory | Fabrizio G.,G Protein mediated Signalling Laboratory | Scarpa E.S.,G Protein mediated Signalling Laboratory | Di Paola S.,G Protein mediated Signalling Laboratory | Di Paola S.,Telethon Institute of Genetics and Medicine
Current Topics in Medicinal Chemistry | Year: 2013

The post-translational modifications of proteins by mono- and poly-ADP-ribosylation involve the cleavage of βNAD+, with the release of its nicotinamide moiety, accompanied by the transfer of a single (mono) or several (poly) ADP-ribose molecules from βNAD+ to a specific amino-acid residue of various cellular proteins. Thus, both mono- and poly-ADP-ribosylation are NAD+-consuming reactions. ADP-ribosylation reactions have been reported to have important roles in the nucleus, and in mitochondrial activity. Distinct subcellular NAD+ pools have been identified, not only in the nucleus and the mitochondria, but also in the endoplasmic reticulum and peroxisomes. Recent reports have shed new light on the correlation between NAD+-dependent ADP-ribosylation reactions and the endoplasmic reticulum. We have demonstrated that ARTD15/PARP16 is a novel mono-ADP-ribosyltransferase with a new intracellular location, as it is associated with the endoplasmic reticulum. The endoplasmic reticulum is a membranous network of tubules, vesicles, and cisternae that are interconnected in the cytoplasm of eukaryotic cells. This intracellular compartment is responsible for many cellular functions, including facilitation of protein folding and assembly, biosynthesis of lipids, storage of intracellular Ca2+, and transport of proteins. ARTD15 might have a role in both the nucleo-cytoplasmic shuttling, through importinβ1 mono-ADP-ribosylation, and in the unfolded protein response through its ability to ADP-ribosylate two components of this pathway: PERK and IRE1. This review summarizes our present knowledge of the enzymes and targets involved in ADP-ribosylation reactions, with special regard to the novel regulatory reactions that occurs at the level of the endoplasmic reticulum, and that can affect the function of this organelle. © 2013 Bentham Science Publishers.


Dani N.,G Protein Mediated Signalling Laboratory | Dani N.,Bioclarma srl | Moura Barbosa A.J.,University of Bologna | Del Rio A.,University of Bologna | Di Girolamo M.,G Protein Mediated Signalling Laboratory
Current Pharmaceutical Design | Year: 2013

Post-translational modifications of cellular proteins by mono- or poly-ADP-ribosylation are associated with numerous cellular processes. ADP-ribosylation reactions are important in the nucleus, and in mitochondrial activity, stress response signaling, intracellular trafficking, and cell senescence and apoptosis decisions. These reversible reactions add ADP-ribose to target proteins via specific enzymes to form the ADP-ribosylated protein; the cleaveage of this covalent bond is performed via hydrolases. Deficiencies in these enzymatic activities lead to cell death or tumor formation, thus defining their functional roles and impact on human disease. Unlike mono- ADP-ribosyltransferases, poly-ADP-ribose polymerases (PARPs) have been at the frontline of drug discovery since the 1980s. PARP1 is a valuable therapeutic target, with a central role in responses to DNA damage. With mono-ADP-ribosylation now linked to human diseases, such as inflammation, diabetes, neurodegeneration and cancer metastasis, novel and equally important functions of mono-ADPribosylation in cell signaling pathways can now be defined. Recently, we reported mono-ADP-ribosylation of ADP-ribosylation factor 6 (ARF6), a small G-protein of the Ras superfamily. In addition to its involvement in actin remodeling, plasma membrane reorganization and vesicular transport, ARF6 contributes to cancer progression through activation of cell motility and invasion. Consequently, targeting this modification will counteract the pro-invasive effects of ARF6, providing innovative anti-tumor therapy. This review summarizes our present knowledge of the enzymes and targets involved in ADP-ribosylation reactions, and describes in silico approaches to visualize their site of interaction and to identify the precise site for ADP-ribosylation. This should ultimately improve pharmacological strategies to enhance both anti-tumor efficacy and treatment of a number of inflammatory and neurodegenerative disorders. © 2013 Bentham Science Publishers.


Dani N.,G Protein mediated Signalling Laboratory | Mayo E.,G Protein mediated Signalling Laboratory | Stilla A.,G Protein mediated Signalling Laboratory | Marchegiani A.,G Protein mediated Signalling Laboratory | And 4 more authors.
Journal of Biological Chemistry | Year: 2011

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ 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 Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ 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 Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ 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 Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Di Girolamo M.,G Protein Mediated Signalling Laboratory
Current Topics in Microbiology and Immunology | Year: 2014

Post-translational modifications of a cellular protein by mono- and poly- ADP-ribosylation involve the cleavage of NAD+, with the release of its nicotinamide moiety. This is accompanied by the transfer of a single (mono-) or several (poly-) ADP-ribose molecules from NAD+ to a specific amino-acid residue of the protein. Recent reports have shed new light on the correlation between NAD+- dependent ADP-ribosylation reactions and the endoplasmic reticulum, in addition to the well-documented roles of these reactions in the nucleus and mitochondria. We have demonstrated that ARTD15/PARP16 is a novel mono-ADP-ribosyltransferase with a new intracellular location, as it is associated with the endoplasmic reticulum. The endoplasmic reticulum, which is a membranous network of interconnected tubules and cisternae, is responsible for specialised cellular functions, including protein folding and protein transport. Maintenance of specialised cellular functions requires the correct flow of information between separate organelles that is made possible through the nucleocytoplasmic trafficking of proteins. ARTD15 appears to have a role in nucleocytoplasmic shuttling, through karyopherin-β1 mono- ADP-ribosylation. This is in line with the emerging role of ADP-ribosylation in the regulation of intracellular trafficking of cellular proteins. Indeed, other,ADPribosyltransferases like ARTD1/PARP1, have been reported to regulate nucleocytoplasmic trafficking of crucial proteins, including p53 and NF-KB, and as a consequence, to modulate the subcellular localisation of these proteins under both physiological and pathological conditions. © Springer International Publishing Switzerland 2014.


PubMed | G Protein mediated Signalling Laboratory
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2011

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.

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