Modulation of Sirtuin NAD+‐dependent Deacylase Activity by Cysteine Oxidation
Authors: Kalous Kelsey S. , Wynia‐Smith Sarah L. , Olp Michael D. , Smith Brian C. 
Published: November 2023

The sirtuin family of proteins catalyze the NAD+‐dependent deacylation of acyl‐lysine residues, producing O‐acyl‐ADP ribose and nicotinamide in the process. Humans encode seven sirtuins (Sirt1‐7) that are considered pro‐survival proteins, as a decrease in sirtuin activity is implicated in several aging‐related diseases including neurodegeneration, cardiovascular disease, and type II diabetes. Consequentially, the pharmaceutical industry is intensely interested in developing sirtuin small‐molecule activators. To determine pathways to increase cellular sirtuin activity with small molecules, we desire to define physiologically relevant mechanisms that decrease sirtuin activity. Elucidation of these mechanisms will provide novel and unexploited means to prevent aging‐associated decreases in sirtuin activity, with the goal of reversing associated pathologies. As cellular oxidative stress is known to increase with age, it remains possible that post‐translational modification of sirtuins by cellular oxidants decreases deacetylase activity.

Indeed, previous studies have shown post‐translational modification of Sirt1 by cysteine S‐nitrosation correlates with increased acetylation of the Sirt1 deacetylase substrates PGC1‐α, p53, and p65. However, the mechanism of sirtuin inhibition by S‐nitrosation was unknown. Here, we show Sirt1 readily undergoes transnitrosation reactions with the physiologically relevant nitrosothiol S‐nitrosoglutathione (GSNO). Steady‐state kinetic analyses and binding assays were consistent with Sirt1 S‐nitrosation inhibiting binding of both the NAD+ and acetyl‐lysine substrates. Additionally, Sirt1 S‐nitrosation correlated with Zn2+ release and selective loss of α‐helical structure without overall destabilization of the enzyme, suggesting the target of S‐nitrosation is the Zn2+‐tetrathiolate subdomain conserved among all sirtuins. Molecular dynamics simulations suggested 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. Furthermore, 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, and this restoration was dependent on the presence of excess free Zn2+, consistent with S‐nitrosation of the Zn2+‐tetrathiolate as the primary source of Sirt1 inhibition. In addition, Sirt6, a second nuclear sirtuin isoform, is readily transnitrosated by GSNO. Furthermore, Sirt6 S‐nitrosation correlates with a decrease in the overall rate of catalysis by Sirt6. These data suggest S‐nitrosation is a widespread physiologically relevant and reversible mechanism to regulate sirtuin activity in vivo. Studies are in progress to determine if S‐nitrosation and other cysteine oxidations modulate the deacylase activity of the remaining five human sirtuins (Sirt2‐5, Sirt7), and to elucidate the physiological or pathological conditions resulting in sirtuin oxidation.

Support or Funding Information

American Heart Association Scientist Development Grant 15SDG25830057 Medical College of Wisconsin Research Affairs Committee American Cancer Society Institutional Research Grants #14‐247‐29‐IRG and #86‐004‐26‐IRG Advancing a Healthier Wisconsin Endowment