title: Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox.
abstract: 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 gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). 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 (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.
authors.affiliation: Neuropharmacology Section, Laboratory of Toxicology & Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Shih-heng
authors.initials: SH
authors.lastname: Chen
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Belinda
authors.initials: B
authors.lastname: Wilson
authors.forename: Jau-shyong
authors.initials: JS
authors.lastname: Hong
authors.forename: Hui-Ming
authors.initials: HM
authors.lastname: Gao
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D003573:Cytochrome b Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects*; D020536:Enzyme Activators / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000201:enzymology / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D011916:Rats, Inbred F344; D012402:Rotenone / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein
chemical_list: D003573:Cytochrome b Group; D020536:Enzyme Activators; D008562:Membrane Glycoproteins; D009479:Neuropeptides; D021122:Protein Subunits; C484602:Rac1 protein, mouse; D012402:Rotenone; D013481:Superoxides; C036888:cytochrome b558; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein
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