NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action

Authors

  • Vincent Jaquet,

    Corresponding author
    1. Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland
      Vincent Jaquet, Deparment of Pathology and Immunology, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail: vincent.jaquet@unige.ch
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  • Julien Marcoux,

    1. Laboratoire des Protéines Membranaires CEA, DSV, Institut de Biologie Structurale (IBS), Grenoble, France
    2. Laboratoire de Spectrométrie de Masse des Protéines, CEA, DSV, Institut de Biologie Structurale (IBS), Grenoble, France
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  • Eric Forest,

    1. Laboratoire de Spectrométrie de Masse des Protéines, CEA, DSV, Institut de Biologie Structurale (IBS), Grenoble, France
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  • Kevin G Leidal,

    1. Inflammation Program and Department of Medicine, Roy J and Lucille A. Carver College of Medicine, University of Iowa, Coralville, IA and the Veterans Administration Medical Center, Iowa City, IA, USA
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  • Sally McCormick,

    1. Inflammation Program and Department of Medicine, Roy J and Lucille A. Carver College of Medicine, University of Iowa, Coralville, IA and the Veterans Administration Medical Center, Iowa City, IA, USA
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  • Yvonne Westermaier,

    1. Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
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  • Remo Perozzo,

    1. Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
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  • Olivier Plastre,

    1. Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland
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  • Laetitia Fioraso-Cartier,

    1. Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland
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  • Becky Diebold,

    1. Department of Pathology, Emory University, Atlanta, GA, USA
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  • Leonardo Scapozza,

    1. Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
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  • William M Nauseef,

    1. Inflammation Program and Department of Medicine, Roy J and Lucille A. Carver College of Medicine, University of Iowa, Coralville, IA and the Veterans Administration Medical Center, Iowa City, IA, USA
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  • Franck Fieschi,

    1. Membrane & Pathogens Group, Institut de Biologie Structurale (IBS), Université Joseph Fourier and Institut Universitaire de France, Grenoble, France
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  • Karl-Heinz Krause,

    1. Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland
    2. Department of Genetic and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland
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    • These authors contributed equally.

  • Karen Bedard

    1. Department of Pathology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada
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    • These authors contributed equally.


Vincent Jaquet, Deparment of Pathology and Immunology, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail: vincent.jaquet@unige.ch

Abstract

BACKGROUND Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood.

EXPERIMENTAL APPROACH Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins.

KEY RESULTS Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration–response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47phox translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47phox and disrupted the binding of the proline rich region of p22phox to the tandem SH3 domain of p47phox and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively.

CONCLUSIONS AND IMPLICATIONS These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

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