Structure and function of poly ADP-ribose glycohydrolases

Abstract

Post-translational modification of proteins by poly ADP-ribosylation is involved in numerous cellular processes such as chromatin restructuring, cell cycle progression and diversion, transcription, DNA repair, cell signalling, apoptosis, necrosis, replicative ageing and wound healing 1-8. As observed for other posttranslational modifications the process is tightly regulated, with proteins functioning as 'readers', 'writers' and 'erasers' coordinating the process.9 A superfamily of enzymes, known as poly (ADP-ribose) polymerases (PARPs), catalyse ADP-ribosylation of target proteins as well as the consequent elongation of poly (ADP-ribose) (PAR) by transferring the ADP-ribose moiety from NAD+. Poly ADP-ribose glycohydrolase (PARG) on the other hand, catalyses the breakdown of PAR into predominantly ADP-ribose monomers10-14. Until recently, research has primarily focused on generation of PAR, with the degradation pathway being comparatively less studied. The recent structure elucidation of a bacterial PARG from T. curvata proved a breakthrough in this field, leading to a postulated model for PARG catalysis in general. Results from our further work with a mutant bacterial PARG showed a role for the G114 residue in directing ligand binding and preventing FAD binding. The structure of PARG from T. curvata was found to be structurally homologous to the MACROD2 protein from H. sapiens, both of which can bind ADP-ribose and both have a slightly different catalytic loop which is responsible for their catalytic activity. Our results suggest that these two loops are not interchangeable, suggesting that the stability and activity of these catalytic loops are governed by the residues surrounding them. We here also present the crystal structure of a canonical PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Our structure reveals that PARG acts predominantly as an exo-glycohydrolase and expands our understanding of the mechanism of poly-ADP-ribose degradation. Although various canonical PARG structures have been reported recently 11,13,15-18, little insight is available for the structure and function of the large regulatory domain present in vertebrate PARGs. Here we present purification of the human regularity PARG region from E. coli as well as initial crystallisation conditions. These results will support further studies in unravelling the complete mechanism of vertebrate PARG. Finally, ADP-ribosylation is more widespread than initially thought, ranging from bacteria including many virulent species, though to humans19. A previously unrecognised class of sirtuins has been discovered in microbial pathogens that possess ADP-ribosylation activity20. The sirtuin-mediated ADP-ribosylation seen in Staphylococcus aureus and Streptococcus pyogenes is dependent on lipoylation, and appears important for the response of these microbial pathogens to their host defence mechanism- oxidative stress20. We present ligand free crystal structure of SAV0323, a bacterial luciferase-like enzyme that is implicated in this response. The structure contains many disordered loops, suggesting FMNH2 and /or substrate binding are required to order the active site region.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)