Detecting Protein Sulfenylation in Human Airway Epithelial Cells (HAEC) Exposed to an Environmental Peroxides
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Exposure to airborne fine particulate matter (PM2.5) is a leading cause of morbidity and mortality worldwide. Exposure to PM2.5 is known to cause a range of pathological complications such as the onset of cardiovascular disease, respiratory infections, and premature death. The largest source of PM2.5 is derived from the oxidation of isoprene by hydroxy radical to form isoprene hydroxy hydroperoxide (ISOPOOH) in the atmosphere. The atmospheric chemistry of ISOPOOH formation is well understood, however, relatively little is known about the adverse human health effects of ambient ISOPOOH exposure. We previously have demonstrated that ISOPOOH exposure induces oxidative stress in HAEC by inducing glutathione oxidation independently of the production of intracellular H2O2. By knocking down the expression of GPx4, we ablated ISOPOOH induced glutathione oxidation, thereby implicating GPx4 involvement specifically. We also demonstrated that ISOPOOH exposure of cellular and acellular membranes induces lipid peroxidation which can be modulated by supplementation with Omega-3 fatty acids. Our current hypothesis is that, in addition to ISOPOOH induced glutathione oxidation, ISOPOOH exposure of HAEC leads to protein sulfenylation. Specifically, GAPDH sulfenylation which, in turn, leads to shunting of glucose to the pentose phosphate pathway. Our experimental approach primarily relies upon dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition reactions to tag intracellular protein sulfenic acids produced by the oxidation of cysteinyl thiols in GAPDH by low micromolar concentrations of H2O2 or ISOPOOH exposure. Exposure of GAPDH to H2O2 or ISOPOOH in the presence of glutathione led to glutathionylation of GAPDH. Tert-butyl hydroperoxide, another low molecular weight hydroperoxide, also induced protein sulfenylation and glutathionylation of GAPDH at low micromolar concentrations. Using a fluorescence imaging approach to detect protein sulfenylation in HAEC, we were able to demonstrate that ISOPOOH exposure led to intracellular protein sulfenylation. These findings demonstrate that ISOPOOH is a potent environmental hydroperoxide capable of inducing oxidative stress through multiple mechanisms involving glutathione oxidation, lipid peroxidation, and sulfenylation of regulatory proteins in HAEC.