title: Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.
abstract: 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.
authors.affiliation: From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Kelsey S
authors.initials: KS
authors.lastname: Kalous
authors.affiliation: From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Wynia-Smith
authors.affiliation: From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Olp
authors.affiliation: From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 brismith@mcw.edu.
authors.forename: Brian C
authors.identifier: http://orcid.org/0000-0001-6330-2768
authors.initials: BC
authors.lastname: Smith
mesh_terms: D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D015538:Nitrosation; D026422:S-Nitrosoglutathione / Q000737:chemistry*; D056564:Sirtuin 1 / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009243:NAD; D026422:S-Nitrosoglutathione; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D015032:Zinc; D003545:Cysteine
keywords: acetylation; allosteric regulation; enzyme inactivation; inhibition mechanism; nicotinamide adenine dinucleotide (NAD); nitric oxide; nitrosation; nitrosylation; sirtuin 1 (SIRT1); zinc