Defining the Biochemical Function and Therapeutic Utility of Unique PARP14 and PARP15 ADP-Ribosylation Sites - SUMMARY ADP-ribosylation was one of the first post-translational modifications (PTMs) discovered. It is a widespread and ubiquitous post-translational modification across all kingdoms of life – whose biochemical selectivity, cellular consequences, and final fates remain poorly understood. Our objective is to investigate the entire life-cycle of the ADP-ribose modification; from ADP-ribose transfer, to the post-transfer alterations of that ADP-ribose, and the final removal of the resulting PTMs. We will use our findings to investigate the functions of the ADP-ribose signal transduction network and develop first-in-class peptide-based inhibitors to target malfunctioning ADP- ribose signaling networks. In humans, 17 different poly-ADP-ribose polymerases (PARPs) catalyze ADP- ribosylation, a separate set of enzymes convert ADP-ribose to phosphoribose, and 6 glycohydrolases remove ADP-ribose. Importantly, there are a multitude of glycohydrolases found in various viral species. The enzymes associated with ADP-ribose processing have been implicated in a variety of physiological functions (e.g., DNA repair, RNA regulation, cell development) and a broad array of diseases, most notably viral infection. While previous efforts have validated the roles for PARPs 14 and 15 in viral regulation, their mechanism of action remains unclear. In large part, this lack of clarity arises from our inability to connect a single site of ADP- ribosylation to its downstream function. Work in the kinase field has demonstrated the power of connecting enzyme-specific PTMs to their precise cellular function, fundamentally altering our understanding of signal transduction and revealing new avenues for therapeutic intervention in disease. By leveraging the expertise from my lab and that of my collaborators, our work will use a multidisciplinary approach to determine the functional consequences of unique ADP-ribosylation events in the cell and develop novel viral glycohydrolase-selective peptide-based inhibitors to block specific ADP-ribose removals. In the first aim, we will identify the auto-modification sites on PARP14 and PARP15, linking specific ADP-ribosylation sites to cellular effects. We have extensive experience working with these PARPs and we have a robust viral model to interrogate how specific site alterations change pathway behavior. In the second aim, a PARP14-specific target peptide sequence that we have developed will serve as a platform to identify selective viral glycohydrolase inhibitors. We will synthesize peptide and peptoid (poly-N-substituted glycine) derivatives of the initial target peptide and identify structure activity relationships (SAR analysis) with the resulting library to optimize promising antivirals. In the third aim, we will map the biochemical pathway responsible for converting ADP-ribose to phosphoribose. We will identify the phosphoribose interactome and investige its role in the innate antiviral response. This research will enhance our mechanistic understanding of ADP-ribosylation and offer new strategies for targeting viral glycohydrolase enzymes, with broader implications for studying PARPs, glycohydrolases, and ADP-ribose processing in medicine and cell biology.
