The Sir2 proteins, also known as sirtuins, comprise a unique family ofprotein deacetylases that are required for diverse biological processes including transcriptional silencing, regulation of fat mobilization, control of the p53 tumor suppressor, and neuronal degeneration. Sir2 proteins also regulate lifespan in yeast, worms and flies; deletion of Sir2 shortens lifespan, while overexpression or stimulation of enzymatic activity extends lifespan. Most sirtuins deacetylate lysine side chains in a unique reaction that consumes NAD+, releasing nicotinamide, O-acetyl ADP ribose, and the deacetylated substrate. The nicotinamide reaction product is a non-competitive inhibitor of some sirtuins and plays an important role in vivo in regulating Sir2 activity. Recent studies show that some sirtuins ADP-ribosylate protein substrates. The outstanding questions in Sir2 biology center on the nature of the unusual enzyme mechanism of these enzymes and how the NAD+ cleavage reaction is altered in some sirtuins to tip the balance towards ADP-ribosylation, rather than NAD+-dependent deacetylation. Structural studies will be carried out on bacterial, archaeal and mammalian sirtuins aimed at elucidating the structure-based enzymatic mechanism. The research will focus on sirtuins with either deacetylase or ADP ribosyltransferase activity in order to determine the structural basis for dual ADP-ribosylation and deacetylase activities exhibited by some sirtuins. In parallel, a proteomics approach will be used to identify the side chain targets of ADP-ribosylation. Further biochemical studies will explore the precise substrate requirements for the ADP-ribosylation reaction, including whether the substrate needs to contain posttranslational modifications. Together, these structural and biochemical approaches will elucidate the novel chemistry of the fascinating and important Sir2 family of enzymes.
BROADER IMPACT OF THE PROPOSED WORK: The PI runs an active laboratory with graduate students and undergraduate researchers. The research will be carried out exclusively by graduate students and will provide excellent training in using an integrated approach that applies the tools of x-ray crystallography and solution biochemistry to important biological problems. It will also provide support to a minority students.
