Regulation of Mitochondrial Sirtuins Sirt3 and Sirt5 by Small Molecules
Sirtuins are a highly conserved family of protein deacylases that are important regulators of metabolism, longevity and aged-related diseases. There are seven sirtuin isoforms in mammals with different subcellular localization, substrates and biological functions. Three sirtuin isoforms, Sirt3-5, are located in the mitochondria and play significant roles in all mitochondrial processes. This study investigates the modulations of small molecule compounds on mitochondrial sirtuins Sirt3 and Sirt5 using structural characterization as well as biochemical and biophysical analysis. Resveratrol, a polyphenol found in red wine, was reported to activate Sirt1. Testing resveratrol and its related compounds including piceatannol, polydatin, 4’-bromo-resveratrol against Sirt3 showed an inhibitory effect. Among these compounds, brRESV is the most potent Sirt3 inhibitor. Crystal structures of Sirt3 in complex with FdL-1 peptide and piceatannol/polydatin showed a direct interaction between the compounds and the peptide coumarin ring to induce non-productive substrate binding, thus inhibit the enzyme activity. Crystal structures of the complex Sirt3/FdL-1/4’-bromo-resveratrol and Sirt3/ACS2/4’-bromo-resveratrol revealed two different compound binding sites. Biochemical and binding data indicated that the binding site of 4’-bromo-resveratrol in the FdL-1 complex involved in inhibition mechanism whereas the compound binding site in the ACS2 complex might imply the site for the activation mechanism of resveratrol on Sirt1. Resveratrol unrelated compounds including SRT1720, a potent synthetic Sirt1 activator, and Ex-527, a potent Sirt1 inhibitor, also inhibited Sirt3. In the crystal structure of the complex Sirt3/carba-NAD+/SRT1720, the compound showed competition with substrate peptide by occupying the binding region of acetyl lysine. SRT1720 interacts with NAD+ and the binding analysis indicated that the NAM moiety of NAD+ is essential for SRT1720 binding. The crystal structure of Sirt3 in the presence of NAD+ and SRT1720 only showed the ADP-ribose part implying the hydrolysis of NAD+ and the importance of NAM moiety in SRT1720 binding. In contrast to the SRT1720 inhibition mechanism, Ex-527 stabilized a closed sirtuin conformation and prevented the product 2'-O-acetyl-ADP-ribose release. Crystal structure of the complex of Sirt3/native O-alkylamidate intermediate provided more information about the deacetylation reaction.
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