Sirtuins are a family of enzymes which have been linked to inflammation and aging if their activity is chronically depressed. These enzymes catalyze the NAD+-dependent deacylation of acyl-lysine residues, producing O-acyl-ADP-ribose and nicotinamide. Humans encode seven sirtuins (Sirt1-7) that are considered pro-survival proteins. Exactly how sirtuin activity is inhibited during inflammation and aging is largely unknown. We seek to define the physiological mechanisms regulating sirtuin activity and develop chemical tools and probes to monitor their activity.
Sirtuin Inhibition by Peroxynitrite
We have investigated how the aging-related oxidant peroxynitrite (ONOO⁻), a nitric oxide-derived reactive species, post-translationally modifies and inhibits the activity of human sirtuins (Bohl et al, 2024). While sirtuins have long been considered protective against aging-related diseases, the specific mechanisms that impair their function during aging remain unclear. Through comparative analysis, our research demonstrates that ONOO⁻ inhibits the deacylase activity of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 primarily through tyrosine nitration, with mass spectrometry revealing novel nitration sites within their catalytic domains. Additionally, SIRT1 and SIRT6 undergo cysteine sulfenylation, and SIRT6 uniquely forms disulfide bonds via a conserved cysteine (C18), not found in other sirtuin isoforms. These findings highlight that ONOO⁻-mediated oxidative modifications—such as nitration, sulfenylation, and disulfide formation—contribute to sirtuin inhibition and may underlie increased vulnerability to age-related diseases, offering new insight into the molecular connections between oxidative stress and aging.
Activity Based Sirtuin Probes
We have developed activity-based probes targeting Sirt1 in both in vitro and cellular assays (Goetz et al, 2020). These probes contain 4 key elements: 1) thioacetyl lysine for mechanism-based affinity, targeting only active sirtuins; 2) peptide sequences specific for Sirt1; 3) a diazirine motif for covalent labeling upon UV irradiation; and 4) an alkyne for bioorthogonal conjugation to a fluorophore for in-gel detection. We found this activity based probe detected Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches.
Oxidative Post-translational Modification of Sirtuins
It is known oxidative stress increases with age and inflammation. We have sought to define the effects of cellular oxidants on the regulation of sirtuin activity. We demonstrated that Sirt1 can be post-translationally modified by cystein S-nitrosation by the small molecule S-nitrosoglutathione (Kalous et al, 2016). Sirt1 S-nitrosation correlated with Zn2+-release and loss of α-helical structure, suggesting the target of nitrosation is the Zn2+-tetrathiolate domain conserved among sirtuins. Sirt1 S-nitrosation was reversed upon exposure to thiol-based reducing-agents, resulting in restoration of Sirt1 activity. This restoration was dependent on the presence of Zn2+, consistent with Zn2+-tetrathiolate nitrosation as the source of Sirt1 inhibition. A follow up study broadened the scope to include other Sirtuin family members (including Sirt1, 2, 3, 5, and 6) to examine their susceptibility to S-nitrosation and other oxidative post-translational modifications (Kalous et al, 2020). Our review (Kalous et al, 2021) highlighted the state of the knowledge of the field regarding oxidative modifications of sirtuins.
SELECTED REFERENCES
Naatz A, Bohl KS, Jones Lipinski RA, Nord JA, Gehant AL, Hansen PA, Smith BC, and Corbett JA. Role of SIRT3 in the regulation of Gadd45α expression and DNA repair in β-cells. Journal of Biological Chemistry (2025). doi: 10.1016/j.jbc.2025.108451. PMID: 40147772.
Bohl K, Wynia-Smith SL, Jones Lipinski RA, Smith BC. Inhibition of Sirtuin Deacylase Activity by Peroxynitrite. Biochemistry. (2024). doi: 10.1021/acs.biochem.4c00257. PMID: 39256054.
Bursch KL, Goetz CJ, Smith BC. Current Trends in Sirtuin Activator and Inhibitor Development. Molecules (2024) doi:10.3390/molecules29051185.
Kalous KS, Wynia-Smith SL, Smith BC. Sirtuin oxidative post-translational modifications. Front Physiol. 2021. DOI:10.3389/fphys.2021.763417 PMID: 34899389. PMCID: PMC8652059
Goetz CJ, Sprague DJ, Smith BC. “Development of activity-based probes for the protein deacylase Sirt1.” Bioorganic Chemistry. 2020, 104:104232. PMID: 32911193.
Kalous KS, Wynia-Smith SL, Summers SB, Smith BC. “Human sirtuins are differentially sensitive to nitrosating agents and other cysteine oxidants.” Journal of Biological Chemistry. 2020 [epub ahead of print] doi: 10.1074/jbc.RA119.011988. PMID 32371394
Kalous KS, Wynia-Smith SL, Olp MD, Smith BC. “Mechanism of Sirt1 NAD+-dependent Deacetylase Inhibition by Cysteine S-nitrosation”. Journal of Biological Chemistry. 2016, 291, 25398-410.
Smith BC, Settles B, Hallows WC, Craven MW, Denu JM. “SIRT3 substrate specificity determined by peptide arrays and machine learning”. ACS Chemical Biology. 2011, 6, 146–57.
Smith BC, Hallows WC, Denu JM. “A continuous microplate assay for sirtuins and nicotinamide-producing enzymes”. Analytical Biochemistry. 2009, 394, 101–9.
Smith BC and Denu JM. “Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide”. Biochemistry. 2007, 46, 14478–86.
Smith BC and Denu JM. “Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage”. Journal of the American Chemical Society. 2007, 129, 5802–3.
