title: A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1.
abstract: Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.
authors.affiliation: Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ramming
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Okumura
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kanemura
authors.affiliation: Swiss Institutes of Bioinformatics, University of Basel, 4056 Basel, Switzerland; Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Sefer
authors.initials: S
authors.lastname: Baday
authors.affiliation: Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland.
authors.forename: Julia
authors.initials: J
authors.lastname: Birk
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Suzette
authors.initials: S
authors.lastname: Moes
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Spiess
authors.affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Paul
authors.initials: P
authors.lastname: Jenö
authors.affiliation: Swiss Institutes of Bioinformatics, University of Basel, 4056 Basel, Switzerland; Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Bernèche
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Inaba
authors.affiliation: Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland. Electronic address: Christian.Appenzeller@sbl.ch.
authors.forename: Christian
authors.initials: C
authors.lastname: Appenzeller-Herzog
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D049109:Cell Proliferation; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism*; D004721:Endoplasmic Reticulum; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004220:Disulfides; C451899:ERP44 protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D005182:Flavin-Adenine Dinucleotide; D006861:Hydrogen Peroxide; C405327:ERO1A protein, human; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D010100:Oxygen
keywords: Disulfide bond formation; Endoplasmic reticulum; Ero1; Hydrogen peroxide; Oxidative folding; Peroxidase: Free radicals