title: The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.
abstract: Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
authors: From the Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, 60590 Germany, the German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
authors: Kim-Kristin
authors: KK
authors: Prior
authors: the German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany the Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, 60590 Frankfurt am Main, Germany, the Cluster of Excellence "Macromolecular Complexes," Goethe-Universität, 60590 Frankfurt am Main, Germany.
authors: Ilka
authors: I
authors: Wittig
authors: From the Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, 60590 Germany, the German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
authors: Matthias S
authors: MS
authors: Leisegang
authors: the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Jody
authors: J
authors: Groenendyk
authors: the Excellence Cluster Cardio-Pulmonary System, Justus-Liebig-University Member of the German Center for Lung Research (DZL), 60590 Giessen, Germany.
authors: Norbert
authors: N
authors: Weissmann
authors: the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Marek
authors: M
authors: Michalak
authors: the Institute for Biomedical Ageing Research and Center for Molecular Biosciences Innsbruck (CMBI), Universität Innsbruck, 6020 Insbruk, Austria.
authors: Pidder
authors: P
authors: Jansen-Dürr
authors: the King's College London British Heart Foundation Centre, Cardiovascular Division, London WC2R 2LS, United Kingdom, and.
authors: Ajay M
authors: AM
authors: Shah
authors: From the Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, 60590 Germany, the German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany brandes@vrc.uni-frankfurt.de.
authors: Ralf P
authors: RP
authors: Brandes
mesh_terms: D000818:Animals; D037281:Calnexin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D051379:Mice; D018345:Mice, Knockout; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D037281:Calnexin; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human
keywords: NADPH oxidase; blue native PAGE; mass spectrometry (MS); protein-protein interaction; proteomics; reactive oxygen species (ROS)