Sirtuin1 (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post-translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150-250 microM), aldehyde-acrolein (10-30 microM), and cigarette smoke extract (CSE; 0.1-1.5%) in the presence of intracellular glutathione-modulating agents at 1-24 h, and oxidative post-translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin-1 (Glrx1), and wild-type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC(50) of 1% for CSE and 30 microM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.

Peroxiredoxin 6 (Prdx6) exerts its protective role through peroxidase activity against H 2 O 2 and phospholipid hydroperoxides. We hypothesized that targeted disruption of Prdx6 would lead to enhanced susceptibility to cigarette smoke (CS)-mediated lung inflammation and/or emphysema in mouse lung. Prdx6 null (Prdx6 −/− ) mice exposed to acute CS showed no significant increase of inflammatory cell influx or any alterations in lung levels of pro inflammatory cytokines compared to wild-type (WT) mice. Lung levels of antioxidant enzymes were significantly increased in acute CS-exposed Prdx6 −/− compared to WT mice. Overexpressing (Prdx6 +/+ ) mice exposed to acute CS showed significant decrease in lung antioxidant enzymes associated with increased inflammatory response compared to CS-exposed WT mice or air-exposed Prdx6 −/− mice. However, chronic 6 months of CS exposure resulted in increased lung inflammatory response, mean linear intercept (L m ), and alteration in lung mechanical properties in Prdx6 −/− when compared to WT mice exposed to CS. These data show that targeted disruption of Prdx6 does not lead to increased lung inflammatory response but is associated with increased antioxidants, suggesting a critical role of lung Prdx6 and several compensatory mechanisms during acute CSinduced adaptive response, whereas this protection is lost in chronic CS exposure leading to emphysema.

Sirtuin1 (Sirt1) is a class III histone/protein deacetylase that has anti‐aging and anti‐inflammatory properties. We have previously shown that cigarette smoke (CS) decreased Sirt1 protein level in rat lung and human monocyte/macrophages leading to increased NF‐κB dependent pro‐inflammatory cytokine release. As CS contains greater than 1015–17 free radicals, we hypothesized that the cell's redox status influences Sirt1 protein level in response to CS, leading to post‐translational modifications and degradation. Human bronchial epithelial cells (BEAS.2B) were exposed for 24 hrs to different concentrations of cigarette smoke extract (CSE 0.5–1%) in the presence or absence of buthionine sulfoximine (BSO) or N‐acetyl‐L‐cysteine (NAC), an inhibitor and precursor for glutathione biosynthesis respectively. CSE decreased nuclear Sirt1 levels; co‐treatment with BSO further decreased Sirt1, whereas treatment with NAC rescued CSE depletion. Furthermore, CSE caused 4‐hydroxy‐2‐nonenol adduct formation on Sirt1, which was increased by BSO treatment and decreased by NAC. Decreased Sirt1 level was also found to be due to proteasomal degradation as proteasomal inhibitors (ALLN and MG132) attenuated CSE‐mediated degradation of Sirt1. These data suggest that oxidants in CSE modify Sirt1 post‐translationally, marking the protein for proteasomal degradation in lung epithelial cells, which may contribute to the pro‐inflammatory effect of cigarette smoke.Supported by the NIEHS Center Grant ES01247 and Toxicology Training Program Grant ES07026.

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