Disulfide Bond As a Switch for Copper-Zinc Superoxide Dismutase Activity in Asthma

Abstract

Aim: Loss of superoxide dismutase (SOD) activity is a defining biochemical feature of asthma. However, mechanisms for the reduced activity are unknown. We hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation. Result: Activity assays of blood samples from asthmatics and healthy controls revealed impaired dismutase activity of copper-zinc SOD (CuZnSOD) in asthma. CuZnSOD purified from erythrocytes or airway epithelial cells from asthmatic was highly susceptible to oxidative inactivation. Proteomic analyses identified that inactivation was related to oxidation of cysteine 146 (C146), which is usually disulfide bonded to C57. The susceptibility of cysteines pointed to an alteration in protein structure, which is likely related to the loss of disulfide bond. We speculated that a shift to greater intracellular reducing potential might account for the change. Strikingly, measures of reduced and oxidized glutathione confirmed greater reducing intracellular state in asthma, compared with controls. Similarly, greater free thiol in CuZnSOD was confirmed by *2-fold greater N-ethylmaleimide binding to C146 in asthma as compared with controls. Innovation: Greater reducing potential under a chronic inflammatory state of asthma, thus, leads to susceptibility of CuZnSOD to oxidative inactivation due to cleavage of C57-C146 disulfide bond and exposure of usually unavailable cysteines. Conclusion: Vulnerability of CuZnSOD influenced by redox likely amplifies injury and inflammation during acute asthma attacks when reactive oxygen species are explosively generated. Overall, this study identifies a new paradigm for understanding the chemical basis of inflammation, in which redox regulation of thiol availability dictates protein susceptibility to environmental and endogenously generated reactive species. Antioxid. Redox Signal. 18,[412][413][414][415][416][417][418][419][420][421][422][423]

Redox Imaging of Inflammation in Asthma

“…Airway epithelium was obtained by gently brushing the airway, and the freshly obtained cells were immediately processed in the bronchoscopy suite for determination of reduced and oxidized glutathione and reducing potential in human airway epithelium of control subjects and patients with asthma in vivo. Similar to a prior report (4), GSH tended to be higher but GSSG lower in asthma (GSH, mM: patients with asthma, 17 6 3 [n = 3]; control subjects, 8.9 6 0.5 [n = 3]; P = 0.14; GSSG, mM: patients with asthma, 0.38 6 0.04; control subjects, 0.48 6 0.08; P = 0.2), so that the ratio of GSH to GSSG in the airway epithelium of patients with asthma was double that of control subjects (GSH/GSSG: patients with asthma, 43 6 6; control subjects, 20 6 4; P = 0.05) ( Figure 1A). The reduction potential (Eh) of the GSH:GSSG couple calculated by Nernst equation (18,19) revealed a significantly greater intracellular reducing state in the airway epithelium of patients with asthma as compared with control subjects (Eh, mV: patients with asthma, -323 6 4; control subjects, -306 6 3; P = 0.04) ( Figure 1A).…”

“…We have identified that the airway epithelium of patients with asthma is shifted to greater reducing potential, recognized by a higher ratio of reduced to oxidized glutathione (4), which occurs in response to the episodic oxidative inflammation of asthma (1,(5)(6)(7)(8)(9). On this basis, we hypothesized that regional airway inflammation in asthma may be quantitatively evaluated by a nuclear imaging strategy based on uptake of a radiopharmaceutical ( 99m Tc-exametazime [HMPAO]) selectively retained in tissues depending on GSH levels (10-13) (see Figure E1 in the online supplement).…”

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

“…In an interesting recent study in asthma patients, disulfidereduced hSOD1 was elevated in human asthmatic erythrocyte and airway epithelial cells, leading to decreased SOD activity in asthma samples (17). The larger fraction of disulfide-reduced hSOD1 in asthma samples was evidenced by the oxidative modification of a disulfide cysteine (Cys-146) upon treatment with H 2 O 2 .…”

Role of Apo E and superoxide dismutase in patients with obstructive lung diseases

“…19 Another study hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation. 20 An increase in ROS generation in asthma and/or COPD patients is confirmed by the changes in the antioxidant …”

“…Overall, this study identifies a new paradigm for understanding the chemical basis of inflammation. 20 The difference in mean SOD level was found to significantly decrease among asthmatic patients. 25 Antioxidants not only protect against the direct injurious effects of oxidants, but also alter the inflammatory events that play an important role in the pathogenesis of COPD.…”