Detecting Protein Sulfenylation in Human Airway Epithelial Cells (HAEC) Exposed to Environmental Peroxides
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Exposure to airborne fine particulate matter (PM2.5) is a one of the leading causes of global morbidity and mortality worldwide. Exposure to PM2.5 is known to exacerbate respiratory and cardiovascular disease and increased the rate premature deaths. The major source of atmospheric PM2.5 is isoprene hydroxy hydroperoxide that is generated by the photo-oxidation of isoprene by hydroxy radical in the atmosphere. While the atmospheric chemistry of ISOPOOH formation is well characterized, the adverse human health effects of ambient ISOPOOH exposure is poorly understood. We demonstrated previously that ISOPOOH exposure induces oxidative stress in HAEC by promoting intracellular glutathione oxidation. We showed that ISOPOOH-induced glutathione oxidation occurs independently of the production of intracellular H2O2. Furthermore, by knocking down the expression of GPx4 in HAEC, we were able to ablate ISOPOOH induced glutathione oxidation, thereby implicating GPx4 involvement specifically. We then demonstrated that ISOPOOH exposure of cellular and acellular membranes induces lipid peroxidation[SJ1] . 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[SJ2] experimental approach primarily relies upon conjugational methods to detect cysteinyl oxidation using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition reactions to tag intracellular protein sulfenic acids produced by the oxidation of cysteinyl protein thiols (i.e., conversion of -SH to -SOH), specifically in GAPDH, exposed to either H2O2 or ISOPOOH. Additionally, we measure kinetic glycolytic GAPDH activity by measuring the production of NADH in cells and a purified system exposed to either H2O2 or ISOPOOH. Exposure of purified GAPDH to low micromolar concentrations H2O2 or ISOPOOH induced protein sulfenylation. This was accompanied by a loss of acellular GAPDH glycolytic activity following exposure to ISOPOOH. Exposure to HAEC to ISOPOOH increased intracellular levels of protein sulfenylation. Furthermore, the exposure of HAEC to ISOPOOH also resulted in hyper-oxidation of intracellular proteins, specifically sulfonylation (-SO3H). Intracellular GAPDH activity in HAEC exposed to ISOPOOH was dose-dependently inhibited.
These findings demonstrate the exposure to ISOPOOH inhibits glycolytic activity by oxidizing GAPDH in HAEC. This also demonstrates 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.
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