Project Summary/Abstract Over the past decade, sirtuins, the NAD-dependent deacetylases, have emerged as important regulators of metabolism and aging. Numerous papers have shown that sirtuins, and in particular SIRT1, have protective roles against age-related diseases, including type II diabetes, cancer, and neurodegenerative disorders. Also, several studies showed that SIRT1 mediates the beneficial effects of calorie restriction in many metabolic tissues, providing a molecular link between this regimen and its beneficial effects on age-related diseases. These findings were followed by the development of small molecules that act as specific activators of SIRT1, with the hope that they will mimic calorie restriction and be used in treating diseases of aging. However, recent evidence suggests that the activity of sirtuins is reduced during aging likely because of a decline in NAD availability. Thus, investigation of the molecular mechanisms that underline age-dependent reduction in sirtuin activity and the molecular basis of interactions of sirtuins with NAD will guide the development of drugs that enhance sirtuin interaction with NAD and counteract the decline in NAD availability that occurs with aging. Our preliminary data show that yeast SIR2 mutants in lysine K475, which resides in the conserved Rossmann fold domain, rescue SIR2 activity in an NAD deficient strain (npt1) and prolong its replicative lifespan. Preliminary biochemical analysis suggests that the mutants have higher catalytic activity, a characteristic observed also with the same mutations in mammalian SIRT1. In Aim1, we will perform biochemical analyses to investigate the molecular mechanism that underlines the increased catalytic activity of these sirtuin mutants. To further elucidate the mechanism of action of these sirtuin mutants, we plan to solve the structure of this domain bound to NAD, in the context of a shorter but still functional SIR2 protein (mini- SIR2) in collaboration with Cynthia Wolberger. Importantly, the yeast residue K475 is conserved in C. elegans and mammals. In Aim 2, we will determine the effects of SIR2 ortholog mutants in these species, by generating knockin animals, determining their lifespans, and challenging them with various stresses. In Aim 3, we will perform high throughput screen to identify compounds that allow sirtuins to function under low NAD conditions, with the hope that these compounds or their derivatives, will reach the clinic to battle diseases of aging. The screen will be performed in collaboration with the High Throughput facility at the Koch Institute for Integrative Cancer Research. .