A PDI-catalyzed thiol–disulfide switch regulates the production of hydrogen peroxide by human Ero1 |
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| Institution: | 1. Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland;2. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan;3. Swiss Institutes of Bioinformatics, University of Basel, 4056 Basel, Switzerland;4. Biozentrum, University of Basel, 4056 Basel, Switzerland;1. Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morganton, WV 26506, United States;2. C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States;3. Moffitt Cancer Center, Tampa, FL 33612, United States;4. Biochemistry, School of Medicine, West Virginia University, Morgantown, WV 26506, United States;5. Cancer Center, West Virginia University, Morgantown, WV 26506, United States;6. Department of Neuroscience, School of Medicine, West Virginia University, Morgantown, WV 26506, United States;1. Key Laboratory of Luminescence and Real-Time Analytical System (Southwest University), Chongqing Science and Technology Bureau, College of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China;2. College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, China;3. From the Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105,;4. Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland, and;5. Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106 |
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| 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 Cys208–Cys241 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 Cys208/Cys241-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys208/Cys241 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. |
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| Keywords: | Endoplasmic reticulum Hydrogen peroxide Oxidative folding Disulfide bond formation Ero1 Peroxidase: Free radicals |
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