Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and coimmunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated that p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox–gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production.

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► Membrane subunit gp91phox of NADPH oxidase is a novel target for rotenone.
► Rotenone can activate PHOX through a p47phox-independent mechanism.
► p47phox elevated rotenone-mediated PHOX activation.
► Membrane translocation of p67phox is required for rotenone-induced PHOX activation.
► Rac1 enhances p67phox-gp91phox interaction during rotenone-elicited PHOX activation.