84 - Nitro fatty acids as activators of hSIRT6 deacetylase activity

Abstract

Sirtuins are NAD+-dependent deacetylases reported as key factors in metabolism, regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) has been shown in vivo to efficiently deacetylase histone 3, however, in vitro displays low deacetylase activity and possess long-chain fatty acid deacylase activity. So far, only a few compounds have been identified to stimulate the deacetylation activity of SIRT6: long-chain free fatty acids and some polyphenols, specifically quercetin and luteolin, at high concentrations. In this study, recombinant hSIRT6 was expressed and purified from E. coli and zinc content/mole protein was determined. Deacylase activity was measured either using a coupled-enzyme assay (plus nicotinamidase and glutamate dehydrogenase under non-limiting conditions) following NADPH consumption at 340 nm, or by quantifying deacylated peptide product by hplc with time. Synthetic acetylated and myristoylated histone 3 peptides containing tryptophans (H3K9-Ac, H3K9-Myr) were used as substrates. In vitro, hSIRT6 displayed better demyristoilase activity than deacetylase activity; Km for H3K9-Ac > 400 μM while Km = 18 μM for H3K9-Myr. The increase in basal deacetylase activity in the presence of 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. The formation of a Michael adduct with cysteine residues was confirmed. In addition, tertiary structure changes after incubation with the nitroalkene was evidenced by near-UV CD spectra, that is likely responsible for the observed reduction in Km for the acetylated peptide substrate. Nitro oleic acid had no significant effect on the demyristoilase activity, thus appears as an specific activator of hSIRT6 deacetylase activity.