A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1

Thomas Ramming; Masaki Okumura; Shingo Kanemura; Sefer Baday; Julia Birk; Suzette Moes; Martin Spiess; Paul Jenö; Simon Bernèche; Kenji Inaba; Christian Appenzeller-Herzog

doi:10.1016/j.freeradbiomed.2015.02.011

A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1

Thomas Ramming, Masaki Okumura, Shingo Kanemura, Sefer Baday, Julia Birk, Suzette Moes, Martin Spiess, Paul Jenö, Simon Bernèche, Kenji Inaba, Christian Appenzeller-Herzog^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

58 Citations (Scopus)

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.

Original language	English
Pages (from-to)	361-372
Number of pages	12
Journal	Free Radical Biology and Medicine
Volume	83
DOIs	https://doi.org/10.1016/j.freeradbiomed.2015.02.011
Publication status	Published - 19 May 2015
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.

Funding

This paper is dedicated to Emil, who was born during the finalization of the manuscript. We are grateful to Alex Odermatt for generous support. We also thank Adam Lister for editing of the manuscript; Lloyd Ruddock, Neil Bulleid, Jan Riemer, Miklos Geiszt, Roberto Sitia, Hans-Peter Hauri, and Ari Helenius for sharing reagents; and PRACE for awarding us access to resource Lindgren based in Sweden at the PDC Center for High Performance Computing. This work was supported by a Ph.D. fellowship from the Boehringer Ingelheim Fonds (to T.R.), a Grant-in-Aid for JSPS Fellows (to M.O.), a grant for Next Generation World-Leading Researchers from MEXT (to K.I.), and the Swiss National Science Foundation (Ambizione), the University of Basel, and the Freiwillige Akademische Gesellschaft (all to C.A.H.). The synchrotron radiation experiment was performed at BL45XU in SPring-8 with the approval of RIKEN (Proposal No. 2014A1345).

Funders	Funder number
Universität Basel
Freiwillige Akademische Gesellschaft
Boehringer Ingelheim Fonds
Japan Society for the Promotion of Science	15H04335, 15J06349, 13J04030
Ministry of Education, Culture, Sports, Science and Technology
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	144111, 142964

Keywords

Disulfide bond formation
Endoplasmic reticulum
Ero1
Hydrogen peroxide
Oxidative folding
Peroxidase: Free radicals

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10.1016/j.freeradbiomed.2015.02.011

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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 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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author = "Thomas Ramming and Masaki Okumura and Shingo Kanemura and Sefer Baday and Julia Birk and Suzette Moes and Martin Spiess and Paul Jen{\"o} and Simon Bern{\`e}che and Kenji Inaba and Christian Appenzeller-Herzog",

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AU - Ramming, Thomas

AU - Okumura, Masaki

AU - Kanemura, Shingo

AU - Baday, Sefer

AU - Birk, Julia

AU - Moes, Suzette

AU - Spiess, Martin

AU - Jenö, Paul

AU - Bernèche, Simon

AU - Inaba, Kenji

AU - Appenzeller-Herzog, Christian

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AB - 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.

KW - Disulfide bond formation

KW - Endoplasmic reticulum

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KW - Hydrogen peroxide

KW - Oxidative folding

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A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1

Abstract

Bibliographical note

Funding

Keywords

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