Strategies for structural studies of poly(ADP-ribose) glycohydrolase: Towards the validation of a novel therapeutic target
Keywordsnicotinamide adenine dinucleotide
AdvisorJacobson, Myron K.
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PublisherThe University of Arizona.
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EmbargoEmbargo: Release after 5/5/2011
AbstractPoly(ADP-ribosyl)ation is a reversible post-translational modification of histones and nuclear proteins rapidly stimulated by DNA damage. Its homeostasis is a dynamic process regulated by the synthesizing enzymes poly(ADP-ribose) polymerases (PARPs) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). PARP-1, the first-discovered and major PARP, has been the focus of many studies aimed at clarifying the biological function of poly(ADP-ribose) (PAR). This abundant nuclear enzyme plays key roles in a variety of cellular processes, including the regulation of chromatin structure, transcription and genomic integrity. Its multifunctionality has made it an attractive and potential target for therapy, as evidenced by the numerous PARP-1 inhibitors currently undergoing clinical trials. The transient nature of PAR, explained by the close coordination between PARP-1 and PARG, has also highlighted the potential of targeting PARG for diseases of inappropriate cell death. A number of obstacles, however, have prevented PARG from being studied as extensively as PARP-1. The extreme sensitivity of PARG to proteases and its insolubility at high concentrations have limited structure-activity relationship analyses and structural studies of PARG, and the unavailability of high-throughput activity assays has stalled the discovery and development of specific and cell permeable PARG inhibitors, subsequently slowing down the validation of PARG as a therapeutic target. The work presented in this dissertation describes in detail strategies devised to overcome these difficulties. First, a novel colorimetric high-throughput assay for PARG was evaluated and its sensitivity and precision were compared to a widely-used radiolabelling assay. Second, several expression and purification systems were constructed in order to obtain high quantities of soluble human PARG protein adequate for in vitrostructural studies. The efficacy of these strategies was demonstrated in structure-activity analyses of PARG which led to the identification of a regulatory segment far removed linearly from the catalytic site of PARG. This region, necessary for catalytic activity, corresponds with a recently identified mitochondrial targeting sequence (MTS) and was thus named the ‘regulatory segment/MTS’ (REG/MTS). Finally, based on structural data obtained, secondary structure predictions were made to provide insight into the molecular composition of the different domains of PARG, whose structures still remain to be determined.
Degree ProgramGraduate College