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Title: The mammalian ADP-ribosyl proteome and characterisation of novel putative intracellular mono-ADP-ribosyl transferases
Author: Di Paola, Simone
ISNI:       0000 0004 2715 0527
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2011
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Protein mono-ADP-ribosylation is a reversible post-translational modification that has been implicated in the regulation of different cell functions, including signal transduction, protein trafficking, and immune responses. In mammals, three families of proteins catalyse this reaction: the ecto-monoARTs/ARTCs, some members of the PARP/ARTD family and some members of the sirtuin family. In this study, I have been focused on the characterisation of human P ARP 161 ARTD 15 and hamster cARTC2.1. Moreover, I have set up novel technologies for the study of intracellular ADP- ribosylation reactions. I have shown that PARP16/ARTD15 is a mono-ADP-ribosyl transferase that localises to the endoplasmic reticulum, where it binds karyopherin-B 1, a pivotal nuclear transport receptor. Moreover, I have also shown that PARP16/ARTD15 induces the mono-ADP-ribosylation of karyopherin-B 1, defining the first substrate for this enzyme. These data suggest a novel regulatory mechanism of karyopherin-Bl functions mediated by PARP16/ARTD15 mono-ADP-ribosyl transferase activity. In parallel, I have also characterised the activity and the intracellular localisation of the hamster isoform cARTC2.1. This enzyme has both NAD-glycohydrolase (NADase) and arginine-specific ADP-ribosyltransferase (ART) activities. I have generated a double mutant of cARTC2.1 by substituting both the glutamates of the EKE active-site motif and showed that this mutated protein loses ART activity, while correctly localise to the plasma membrane. Importantly, we have shown that endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population and shows higher level of cARTC2.1 transcript compared to the ART- negative population. Moreover, I have demonstrated that cARTC2.1 catalyses the mono-ADP- ribosylation of GRP78/BiP, a protein with a central role in different cell functions. This finding represents the first demonstration of the ARTC-mediated GRP78/BiP modification. Finally, I have set up experimental conditions for GST-macro domain based approaches. I have shown that the cross-linking of GST-macro domain to the sepharose resin improves ADP- ribosylated substrate isolation and analysis by macro-based pull-down assay. Moreover, I have demonstrated the suitability of GST-macro domain to label endogenous ADP-ribosylated proteins by a far-western blotting approach. These data established that GST -macro domain is a powerful tool in the study of ADP-ribosylation reactions at protein level.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available