Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation

• Published in: The Journal of biological chemistry 2016-12, Vol.291 (49), p.25398-25410
• Main Authors: Kalous, Kelsey S., Wynia-Smith, Sarah L., Olp, Michael D., Smith, Brian C.
• Format: Article
• Language: English
• Subjects: acetylation; allosteric regulation; Animals; Cattle; Cysteine - chemistry; Cysteine - metabolism; enzyme inactivation; Enzyme Stability; Enzymology; Humans; inhibition mechanism; NAD - chemistry; NAD - metabolism; nicotinamide adenine dinucleotide (NAD); nitric oxide; Nitrosation; nitrosylation; S-Nitrosoglutathione - chemistry; sirtuin 1 (SIRT1); Sirtuin 1 - chemistry; Sirtuin 1 - metabolism; zinc; Zinc - chemistry; Zinc - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.754655

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1–7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment.