title: Hyperactivity of the Ero1α oxidase elicits endoplasmic reticulum stress but no broad antioxidant response.
abstract: Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.
authors.affiliation: Department of Biology, University of Copenhagen, 2200 Copenhagen N., Denmark.
authors.forename: Henning Gram
authors.initials: HG
authors.lastname: Hansen
authors.forename: Jonas Damgård
authors.initials: JD
authors.lastname: Schmidt
authors.forename: Cecilie Lützen
authors.initials: CL
authors.lastname: Søltoft
authors.forename: Thomas
authors.initials: T
authors.lastname: Ramming
authors.forename: Henrik Marcus
authors.initials: HM
authors.lastname: Geertz-Hansen
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.forename: Esben Skipper
authors.initials: ES
authors.lastname: Sørensen
authors.forename: Agnieszka Sierakowska
authors.initials: AS
authors.lastname: Juncker
authors.forename: Christian
authors.initials: C
authors.lastname: Appenzeller-Herzog
authors.forename: Lars
authors.initials: L
authors.lastname: Ellgaard
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D019943:Amino Acid Substitution; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D056811:Unfolded Protein Response
chemical_list: C465506:CRELD1 protein, human; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D016166:Free Radical Scavengers; D008562:Membrane Glycoproteins; D019328:Buthionine Sulfoximine; C405327:ERO1A protein, human; D010088:Oxidoreductases; D000111:Acetylcysteine