Sirtuins are enzymes which 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. Decreased sirtuin activity promotes inflammation and aging-related diseases. However, how sirtuin activity is inhibited during aging is largely unknown. We seek to define the physiological mechanisms regulating sirtuin activity and develop chemical tools and probes to monitor their activity.

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.


Kalous KS, Wynia-Smith SL, Smith BC. Sirtuin oxidative post-translational modificationsFront 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: . 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.

Scroll to top