Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2 |
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Authors: | Csányi Gábor Cifuentes-Pagano Eugenia Al Ghouleh Imad Ranayhossaini Daniel J Egaña Loreto Lopes Lucia R Jackson Heather M Kelley Eric E Pagano Patrick J |
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Affiliation: | a Vascular Medicine Institute, Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15260, USAb Department of Pharmacology, Biomedical Sciences Institute, University of São Paulo, 05508 900 São Paulo, Brazilc Department of Pathology and Experimental Medicine, Emory University School of Medicine, Atlanta, GA 30322, USAd Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA 15260, USA |
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Abstract: | In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O2•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O2•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent. |
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Keywords: | AngII, angiotensin II COS-22, COS-7 cells transfected with p22phox COS-Nox1, COS-22 cells transfected with Nox1, NOXO1, and NOXA1 COS-Nox2, COS-22 cells transfected with Nox2, p47phox, and p67phox COS-Nox4, COS-22 cells transfected with Nox4 DPI, diphenyleneiodonium chloride DUOX, dual oxidase EPR, electron paramagnetic resonance LiDS, lithium dodecyl sulfate Nox, NADPH oxidase Nox2ds, Nox2 docking sequence NOXA1, Nox activator subunit 1 NOXO1, Nox organizer subunit 1 O2&bull &minus , superoxide anion PMA, phorbol myristate acetate PMSF, phenylmethanesulfonyl fluoride ROS, reactive oxygen species SOD, superoxide dismutase XO, xanthine oxidase X, xanthine |
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