Regulation and function of site-specific protein poly-ADP-ribosylation

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PROJECT SUMMARY Protein ADP-ribosylation (ADPr) is a dynamic, NAD+-dependent post-translational modification. The mammalian poly(ADP-ribose) polymerase (PARP) proteins that catalyze ADPr target several chemically distinct amino acid side chain functionalities on hundreds of substrate proteins to mediate a multitude of orthogonal signal transduction pathways. Adding to this complexity is the potential for ADP-ribose polymer formation, a process wherein the PARP1/2 and TNKS1/2 enzymes elongate ADP-ribose chains from mono-ADPr sites. Highlighting the importance of poly-ADP-ribose in physiology and disease are: (i) the expanding clinical utility of PARP1/2 inhibitors to treat DNA repair-deficient cancers, and (ii) TNKS1/2 function in Wnt/b-catenin signaling and dysfunction in developmental diseases including Cherubism. Aberrant ADPr activity has also been reported as an underlying cause of cardiovascular and neurogenerative diseases, and these findings have inspired intense efforts to elucidate PARP substrate profiles, determine PARP regulatory mechanisms, and develop PARP isoform-specific inhibitors. However, given the liberal deployment of ADPr in cellular signaling and its topologically complex chemical nature, our understanding of how specific mono- and poly-ADPr sites impact protein function and elicit distinct biological activities has lagged behind. The proposed work aims to fill this knowledge gap by developing novel approaches to reconstitute ADPr-mediated signaling events in highly controlled biochemical and cellular environments. We recently developed a chemoenzymatic strategy to install serine ADPr onto peptides and proteins with full control over modification site and ADP-ribose chain length. Using this technology, we identified critical molecular determinants of DNA damage-induced chromatin remodeling and uncovered specialized functions for nucleosome serine poly-ADPr. We are now in a unique position to build upon our technologies and address fundamental questions in PARP biology. We will explore mechanisms that govern poly-ADPr activity and investigate how different modification sites and accompanying polymer lengths encode for specific biochemical outputs throughout the cell. Such information may guide more effective strategies to identify and treat diseases that rely on dysfunctional ADPr activity.