Redox modulation of hSIRT6, key enzyme of metabolism and inflammation

Sirtuins are NAD + -dependent deacetylases reported as key factors in metabolism regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) is known as a histone 3 deacetylase despite of its low activity in vitro . Its deacetylase activity increases in the presence of fatty acids and possess long chain fatty acid deacylase activity. Studies on regulation of hSIRT6 activity are scarce. Our aim was to investigate the potential redox regulation of hSIRT6, characterizing structural and functional changes under different redox conditions. Recombinant hSIRT6 was expressed and purified from E. coli . Deacylase activity was measured using a coupled-enzyme assay (plus nicotinamidase and glutamate dehydrogenase under non-limiting conditions) following NADPH consumption at 340 nm. Synthetic acetylated and myristoylated histone 3 peptides (H3K9-Ac, H3K9-Myr) were used as substrates. Total protein thiols were oxidized after hSIRT6 treatment with H 2 O 2 (50x, 10 min), however, deacetylase activity was not significantly altered, while treatment with an excess of peroxynitrite reduced protein thiol content with a significant loss of activity. The increase in basal deacetylase activity in the presence of 100 μM oleic acid was confirmed. Surprisingly, treatment with the electrophilic derivative nitro-oleic acid do not inactivate, on the contrary, enhanced deacetylase activity, albeit the presence of a catalytic histidine. Other nitro-fatty acids gave the same response. Thermodynamic analysis with determination of dissociation constants is underway.