Biochemical and structural characterization of Sirtuins from mammals and Thermotoga maritima
Sirtuins are NAD+ dependent protein deacetylases involved in regulation of metabolism, age related diseases and are suggested to mediate lifespan extending effects of calorie restriction. Mammals contain seven Sirtuin soforms with various cellular localizations and substrate preferences. This work focuses on the structural and biochemical characterization of several mammalian Sirtuin isoforms, with main emphasis on the largest member of the family, Sirt1.Analysis of various Sirt1 constructs using analytical ultracentrifugation and electron microscopy techniques identified it to be a monomer, similar to other mammalian isoforms. Comparison of activity between different Sirt1 constructs indicates that the termini might be involved in regulating the protein’s activity. It could be shown that the catalytic domain of Sirt1 is sufficient for its activation by the lifespan extending polyphenol resveratrol and that resveratrol can directly bind to Sirt1. Using a peptide array harboring all known mammalian acetylation sites several novel substrates for Sirt1 were identified and shown that resveratrol and other small molecules can modulate Sirt1 dependent deacetylation of substrate peptides and that the effect is specific for the sequence of the substrate site. This finding reveals exciting possibilities for the development of new modulators targeting only specific substrates of Sirt1. These compounds can further be used in analyzing the functions of Sirtuins in vivo and can also serve as attractive leads for developing Sirt1 specific drugs. Studies on the mitochondrial isoform Sirt3 revealed that Zinc is essential for the structural integrity of Sirtuins. Investigations on Sirt5, also a mitochondrial Sirtuin appears to indicate that it is insensitive to nicotinamide (a physiological inhibitor of Sirtuins). Studies on the nucleolar isoform Sirt7 identified Thr224 to be phosphorylated, and play a role in the enzyme’s activity. The thermotoga maritima Sirtuin Sir2Tm was used as a model system for structural characterization of Sirt1. The crystal structure of Sir2Tm in complex with Ex-527, a potent Sirt1 inhibitor was solved, which combined with biophysical studies offers information about Ex-527 binding and inhibition. Close contacts between Ex-527 and some form of ADP-ribose, possibly the product O-acetyl-ADP-ribose (which is not yet fully verified) in the closed enzyme conformation prevent the release of products, thereby stalling the enzymatic reaction. These results offer insights into the isoform specific inhibition of Sirtuins by Ex-527 and provide information for further improvement of Sirt1 specific inhibitors.
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