Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 280, Issue 17, Pages 17187-17195Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M501250200
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- NIGMS NIH HHS [F31 GM066366, R01 GM65386, NIH 5 T32 GM08349] Funding Source: Medline
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The NAD(+)-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested ( yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation (similar to 8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group.
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