Cellular thiol redox environment modulates SIRT1 activity

Sirtuin-1 (SIRT1) is an NAD+-dependent deacetylase that removes an acetyl group from lysine residues of intracellular proteins such as histones. SIRT1 is intimately involved in vascular homeostasis and regulates endothelial cell apoptosis, senescence, and migration. Thus, SIRT1 is a promising therapeutic target in the treatment of diseases that damage the endothelium, such as atherosclerosis, type 2 diabetes, and aging. SIRT1 contains nineteen cysteines, including four cysteines that coordinate a zinc ion to form a structural zinc finger domain that is essential to enzymatic activity. S-glutathiolation is a reversible cysteine thiol modification that regulates protein function. Under pathophysiological conditions, these cysteines may be subject to irreversible oxidative modifications that are detrimental to enzymatic activity and vascular function. The hypotheses explored in this work are first, that SIRT1 activity may decrease upon oxidation of cysteines, and second, SIRT1 stimulation by the polyphenol resveratrol (RSV) is dependent on the reversible oxidation of SIRT1 via S-glutathiolation. In vitro treatment of SIRT1 with S-nitrosoglutathione (GSNO) inhibited RSV stimulation with an IC50 of 69 muM. Overexpression of glutaredoxin-1 or depletion of intracellular GSH with buthionine sulfoximine (BSO) increased ex vivo SIRT1 response to RSV. Moreover, BSO decreased p53 acetylation demonstrating increased intracellular SIRT1 activity. Mass spectrometry indicated that SIRT1 is oxidized by GSNO on five cysteines, including cysteine 67. In addition to S-glutathiolation, which occurs under physiological conditions, reactive cysteine thiols are vulnerable to irreversible oxidative modifications that result from elevated production of ROS/RNS including sulfination (-SO2H) and sulfonation (-SO3H). Unlike glutathiolation, these modifications may not be reversed by cellular processes and are deleterious to protein function. Exposure of SIRT1 to oxidants such as peroxynitrite decreased SIRT1 activity and decreased binding to a zinc-NTA column. Furthermore, treatment of endothelial cells with the peroxynitrite donor, SIN1, or with TNFalpha, decreased biotinylated iodoacetamide (bIAM) labeling of thiolate cysteines. In summary, our evidence indicates for the first time that SIRT1 cysteines are susceptible to oxidative post-translational modification (OPTM) that may become dysregulated under conditions of elevated oxidative stress in the endothelium and inhibit SIRT1 activity. Furthermore, stimulation by the polyphenol, RSV, is attenuated by S-glutathiolation.