GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide

Abstract

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Keywords: Air pollution; Cellular bioenergetics; GAPDH (glyceraldehyde-3-phosphate dehydrogenase); Oxidative stress; Protein thiol oxidation; Secondary organic aerosols.

Graphical abstract

Fig. 1

ISOPOOH induces intracellular protein thiol oxidation. HAEC were exposed to 0 (Vehicle Control) – 1 mM ISOPOOH or H₂O₂ for 10 min, followed by labeling with DAz-2 and conjugation to Avidin-FITC using the Staudinger ligation reaction. Fluorescent intensities were background corrected, and comparisons were performed using a one-way ANOVA with multiple comparisons to vehicle control with Dunnett correction, *p ≤ 0.05. Data shown are representative images of three independent experiments.

Fig. 2

HAEC exposure to ISOPOOH induces intracellular GAPDH thiol oxidation. HAEC were exposed to Vehicle Control, 1 mM ISOPOOH, or 1 mM H₂O₂ for 10 min and labeled with DYn-2 followed by conjugation to biotin by CuAAC. (A) Western blot with three different biological replicates showing protein thiol oxidation between 15 and 150 kD in HAEC exposed to ISOPOOH. (B) The same blot was stripped and reblotted for GAPDH. (C) The same lysates were then subjected to avidin immunoprecipitation and blotted against GAPDH. (D) Densitometry measurements of total protein thiol oxidation shown in A. (E) Densitometry of intracellular GAPDH thiol oxidation shown in C. Quantitations were normalized to background and loading control and compared using a two-tailed unpaired t-test with Welch's correction, *p ≤ 0.05. Data shown are western blots of three independent experiments.

Fig. 3

HAEC exposure to ISOPOOH induces intracellular GAPDH sulfonylation. Proteins from HAEC exposed to 0–1 mM ISOPOOH and 1 mM H₂O₂ for 10 min were blotted for GAPDH-SO₃ and total GAPDH. Densitometry measurements were normalized to background and loading control. Comparisons were performed using a one-way ANOVA with multiple comparisons to vehicle control with Dunnett correction, *p ≤ 0.05. Data shown as representative images of western blots of four independent experiments.

Fig. 4

ISOPOOH induces dose-dependent GAPDH sulfenylation. Rabbit muscle GAPDH was treated with 0 (Vehicle Control) – 100 μM ISOPOOH for 5 min and labeled with dimedone (DMD). Blot was probed with antibodies specific for 2-thiodimedone (2-thio-DMD) and GAPDH. Densitometry measurements were normalized to background and loading control. Comparisons were performed using a one-way ANOVA with multiple comparisons to vehicle control with Dunnett correction, *p ≤ 0.05. Data shown as representative images of western blots of three independent experiments.

Fig. 5

ISOPOOH exposure dose-dependently inhibits GAPDH dehydrogenase activity. Rabbit muscle GAPDH was treated with 0 (Vehicle Control) – 1 mM ISOPOOH, 1 mM DTT (reducing control), or 1 mM H₂O₂ (positive control) for 5 min. GAPDH was diluted in a phosphate buffered solution containing glyceraldehyde-3-phosphate and NAD+. GAPDH activity was measured in a reaction monitored at 340 nm absorbance which marked the formation of NADH. All values are presented as mean ± SEM, n = 4. Comparisons were performed using a repeated measures ANOVA with multiple treatment groups and post-hoc Tukey's multiple comparisons test, *p ≤ 0.05.

Fig. 6

Exposure to ISOPOOH induces dose-dependent inhibition of GAPDH activity in HAEC. GAPDH glycolytic activity was measured at 450 nM absorbance. Lysates prepared from HAEC exposed to 0 (Vehicle Control) – 1 mM ISOPOOH, 1 mM DTT (reducing control), or 1 mM H₂O₂ (positive control) for 10 min were assayed for GAPDH activity, measured as the reduction of NAD + to NADH. GAPDH activity during the linear region of the curves (initial 5 min) was statically lower in HAEC exposed to 1 mM ISOPOOH (0.11 nmol NADH/min) compared to control (0.869 nmol NADH/min). During acquisition, 1 mM DTT was added at the indicated time. All values are presented as mean ± SEM, n = 3. Comparisons were performed using a repeated measures ANOVA with multiple treatment groups and post-hoc Tukey's multiple comparisons test, *p ≤ 0.05.