Investigation of Terminal ADP-ribose protein Glycohydrolase-1 (TARG1) in the DNA damage response and neurodegenerative disease

Abstract

ADP-ribosylation is a post-translational modification of proteins, which has well- established roles in the DNA Damage Response (DDR) and the regulation of chromatin structure. Its monomeric or polymeric addition to proteins is primarily catalysed by the poly(ADP-ribose) (PAR) polymerase (PARP) family of proteins, whose founding member, PARP1, is responsible for the synthesis of roughly 90% of cellular PAR. The last decade has seen significant advances in our understanding of the catabolic regulation of this modification by PAR glycohydrolase (PARG) - the primary protein responsible for its removal. However, a long-standing question in the field has been the identity of the protein or proteins that process the terminal ADP-ribose units of PAR chains, as well as protein mono-ADP-ribosylation - itself a signal of emerging importance. In 2013, pioneering research from our lab and others unveiled a trio of macrodomain-containing proteins that possessed this long-sought activity: MacroD1, MacroD2 and C6orf130, the latter of which was subsequently renamed Terminal ADP-ribose Protein Glycohydrolase- 1 (TARG1), in reflection of its new-found activity. The structural and biochemical insights into TARG1 presented in our paper provide the basis for this thesis, which seeks to translate this biochemical activity into a physiological function. Herein, we describe the first evidence of the proposed TARG1 mechanism occurring in cells, and identify a role for TARG1 in the DDR. We also build on the work of our collaborators in London, who identified an extended family of patients with an inherited mutation in the Targ1 gene, each suffering from a severe form of progressive neurodegeneration. Through RNA- mediated depletion of TARG1, particularly in glioblastoma cells, we identify senescence as a major phenotype emerging after loss of TARG1, but go on to demonstrate pleiotropic effects. We attempt to relate these phenotypes to upstream DNA damage and PAR signalling pathways, and identify the vital role of p53 in mediating the effects of TARG1 depletion. Ultimately, the results presented in this thesis highlight the need for further investigations on a whole-organism scale in order to better understand TARG1 function, particularly in relation to the neurodegenerative disease phenotype.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ivan Ahel (Supervisor) & Ian Waddell (Supervisor)