Sirtuin.

Sirtuin Projects

This research is directed at understanding the function of the Silent Information Regulator 2 (Sir2) family of enzymes, both at the biochemical and cellular level. Recent evidence has indicated that these proteins possess unique enzymatic activities (NAD⁺-dependent protein deacetylation) that are required for their chromatin silencing effects. Evolutionarily conserved, the Sir2 family has been implicated in a wide range of biological activities, including gene silencing, chromosomal stability, metabolic control, and life-span extension via caloric restriction. We have discovered that Sir2 enzymes are potent histone/protein deacetylases that couple protein deacetylation to the production of a completely novel metabolite O-acetyl-ADP-ribose (OAADPr). New evidence suggests that generation of OAADPr may mediate important biological functions. This metabolite may be the key to understanding the diverse biological functions of Sir2 enzymes. For instance, the generation of OAADPr may link gene silencing in the nucleus with proper metabolic control. However, the mechanism of how Sir2 proteins accomplish this reaction and the functional significance of producing OAADPr are not understood. To probe the biological and biochemical functions of this unique family of enzymes, this research provides a fully integrated approach utilizing biochemistry, proteomic methods, biochemical genomics, structure and chemistry to address how and why Sir2 enzymes catalyze this unique reaction and the production of OAADPr. Given the vast data relating chromosomal instability and disease (e.g. cancer) with chromatin remodeling enzymes, our understanding of these molecular mechanisms may lead to the development of rationale therapeutics that inhibit Sir2 enzyme function.

 Catalytic Mechanism and Mechanism-based Inhibitor Design

The lab has made considerable progress toward understanding how proteins are deacetylated in an NAD+-dependent manner. Ongoing studies are addressing transition state structure, the importance and mechanism of Sirtuins reported ADP-ribosylation activity, and the design of mechanism-based inhibitors.

Project Personnel: Brian Smith

Sirtuins catalyze the deacetylation of protein substrates and produce the novel metabolite, OAADPr (O-acetyl-ADP-ribose), which recently has been implicated in binding to proteins involved in transcription repression and silencing, as well as ion transport across the plasma membrane. Evidence suggests that OAADPr may mediate some of the known phenotypic changes associated with sirtuin function. Current studies are aimed at providing direct evidence for the role of OAADPr, and at identifying new protein targets of this unusual metabolite. Yeast and mammalian cultured cells are being utilized to answer these questions.

Project Personnel: Suzi Lee, Lindsay Comstock, Tong Lee

The lab has recently demonstrated that mammalian Acetyl-CoA Synthetases (AceCSs) are regulated by reversible acetylation and that sirtuins activate AceCSs by deacetylation. Site-specific acetylation of mouse AceCS1 on Lys-661 was identified using mass spectrometry and a specific anti-acetyl-AceCS antibody. In mammals, two AceCSs have been identified previously: cytoplasmic AceCS1 and mitochondrial AceCS2. Since SIRT3 is localized to the mitochondria, we investigated whether AceCS2 also might be regulated by acetylation, and specifically deacetylated by mitochondrial SIRT3. AceCS2 was completely inactivated upon acetylation and was rapidly re-activated by SIRT3 deacetylation. Lys-635 of mouse AceCS2 was identified as the targeted residue. Utilizing reversible acetylation to modulate enzyme activity, we have proposed a model for the control of AceCS1 by SIRT1 and of AceCS2 by SIRT3.

Future studies are directed at understanding how this regulatory circuit might be involved in Sirtuin phenotypes associated with aging, neurodegeneration, diabetes, and cancer. In addition, other cellular targets of Sirtuins are being investigated.

Project Personnel: Suzi Lee, Casey Hallows