Abstract
Sirtuins (e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD+-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC50 and kinact/KI analyses, we quantified the ability of nitrosothiols (S-nitrosoglutathione and S-nitroso-N-acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine S-nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S-nitrosation in response to exposure to both free nitric oxide and nitrosothiols (kinact/KI ≥ 5 m-1 s-1), which is the first report of Sirt2 and Sirt6 inhibition by S-nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.
Highlights
Sirtuins catalyze the removal of acetyl and longer acyl-chains from protein lysine residues using NAD1 as a cosubstrate, producing O-acyl-ADP-ribose and nicotinamide [1]
Sirtuin isoforms are differentially inhibited by physiological cysteine oxidants To directly compare the susceptibility of human sirtuins to inhibition by common cellular oxidants, Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6 were treated for 1 h with 100 mM of the nitrosothiols GSNO and SNAP, nitric oxide (NO) (2 mol NO released/mol MAHMA-NONOate, t1/2 5 2.7 min), H2O2, and GSSG
The deacylase activity of treated sirtuins was measured at subsaturating concentrations near the Km values of both acylated peptide and NAD1, either by an enzyme-coupled continuous assay for nicotinamide formation (Sirt1 and Sirt3) [41] or an HPLC fixed time point assay monitoring acylated and deacylated peptide (Sirt2, Sirt5, and Sirt6) [8]
Summary
Sirtuins catalyze the removal of acetyl and longer acyl-chains from protein lysine residues using NAD1 as a cosubstrate, producing O-acyl-ADP-ribose and nicotinamide [1]. Sirt6 (0.5 mM) deacetylase activity was assessed via an HPLC fixed time point assay [8] following incubation with varying concentrations of GSNO (red circles) or NO (2 mol NO released/mol MAHMA-NONOate) (gray triangles) for 0–90 min at 37 °C.
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