PROJECT SUMMARY Type-II diabetes is characterized by the inability of pancreatic beta cells to produce enough insulin to maintain normal levels of blood glucose. In the development of diabetes, prolonged exposure of pancreatic beta cells to macrophage-derived inflammatory cytokines can initiate nitric oxide (NO)-dependent beta cell dysfunction and apoptosis. The sirtuin family (Sirt1-7) of NAD+-dependent protein deacylases are generally considered pro- survival proteins, as decreased sirtuin activity is implicated in the development of several aging-related diseases including type-II diabetes. The nuclear sirtuin Sirt6 is thought to be particularly important in maintaining metabolic health and protecting against pancreatic beta cell dysfunction. The overall goal of this proposal is to elucidate the response of Sirt6 to inflammatory conditions in beta cells. The sensitivity of Sirt6 to posttranslational modification by NO and the impact of Sirt6 modification on beta cell DNA damage repair in response to inflammatory cytokines will be tested in two Specific Aims. In Aim 1, we will test the hypothesis that Sirt6 is S-nitrosated during beta cell inflammation. S-nitrosation is a NO-derived post-translational modification of protein cysteine residues. We and others recently demonstrated that Sirt1 deacetylase activity is reversibly inhibited by S-nitrosation of the Sirt1 Zn2+-tetrathiolate cysteine residues. The Zn2+-tetrathiolate is conserved among all sirtuin isoforms and Sirt6, like Sirt1, is localized to the nucleus. Therefore, Sirt6 may be inhibited by the same NO-dependent pathway as Sirt1. Indeed, our unpublished preliminary data shows that Sirt6 is readily transnitrosated by NO and S-nitrosoglutathione in vitro, and S-nitrosation of Sirt6 correlates with a decrease in Sirt6 deacetylase activity. In Aim 2, we will test the hypothesis that Sirt6 S-nitrosation inhibits DNA damage repair in beta cells. Recent evidence points to a central role for DNA damage in the beta cells that undergo apoptosis in response to cytokine treatment. Among the sirtuins, Sirt6 is uniquely implicated in promoting DNA damage repair. Therefore, we will examine the impact of Sirt6 S-nitrosation in DNA damage repair and beta cell survival. We will use biochemical, biophysical, chemical biological, cell biological, and molecular biological tools and assays to evaluate the regulation of Sirt6 deacetylase activity in beta cells by S- nitrosation and the impact on DNA damage repair processes in beta cells. We anticipate the mechanistic insights gained from this work will improve understanding of type-II diabetes pathogenesis, with the long-term goal of developing novel strategies for prevention or treatment of type-II diabetes.