Proteome-wide Dysregulation by Glucose-6-phosphate Dehydrogenase (G6PD) Reveals a Novel Protective Role for G6PD in Aflatoxin B₁‑Mediated Cytotoxicity

Glucose-6-phosphate dehydrogenase (G6PD) is pivotal to reduced nicotinamide adenine dinucleotide phosphate (NADPH) production and cellular redox balance. Cells with G6PD deficiency are susceptible to oxidant-induced death at high oxidative stress. However, it remains unclear what precise biological processes are affected by G6PD deficiency due to altered cellular redox homeostasis, particularly at low oxidative stress. To further explore the biological role of G6PD, we generated G6PD-knockdown cell clones using lung cancer line A549. We identified proteins differentially expressed in the knockdown clones without the addition of exogenous oxidant by means of isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography–mass spectrometry (LC–MS/MS). We validated a panel of proteins that showed altered expression in G6PD-knockdown clones and were involved in metabolism of xenobiotic and glutathione (GSH) as well as energy metabolism. To determine the physiological relevancy of our findings, we investigated the functional consequence of G6PD depletion in cells treated with a prevalent xenobiotic, aflatoxin B₁ (AFB₁). We found a protective role of G6PD in AFB₁-induced cytotoxicity, possibly via providing NADPH for NADPH oxidase to induce epoxide hydrolase 1 (EPHX1), a xenobiotic-metabolizing enzyme. Collectively, our findings reveal for the first time a proteome-wide dysregulation by G6PD depletion under the condition without exogenous oxidant challenge, and we suggest a novel association of G6PD activity with AFB₁-related xenobiotic metabolism.