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

https://doi.org/10.1016/j.freeradbiomed.2015.02.011Get rights and content

Highlights

  • The regulatory Cys208–Cys241 disulfide in Ero1α is opened by PDI.

  • Ero1α devoid of all regulatory cysteines displays lethal levels of oxidase activity.

  • The Cys208/Cys241 switch regulates O2 access to the active site.

  • Binding of GPx8 to the Cys208/Cys241 region ensures detoxification of produced H2O2.

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.

Introduction

Oxidative protein folding is defined as the assisted process of tertiary structure acquisition of a polypeptide chain, which requires the formation of covalent disulfide crosslinks between specific cysteine side chains. The enzymatic machinery for oxidative protein folding has been extensively described in three subcellular locations: the periplasmic space in gram-negative bacteria [1] as well as the mitochondrial intermembrane space [2] and the endoplasmic reticulum (ER) [3] in eukaryotic cells. In all three compartments, the electrons derived from disulfide-bond formation are transported along specialized biochemical cascades to finally target molecular oxygen (O2) [4]. In the ER, this final step can be catalyzed by the flavoproteins of the ER oxidoreductin 1 (Ero1) family (Ero1α and Ero1β in mammals), which are the best-conserved disulfide-producing enzymes of the ER [5], [6]. The catalytic cycle of Ero1 produces stoichiometric amounts of hydrogen peroxide (H2O2) [7], [8]. Newly generated disulfides are transferred from a flavin adenine dinucleotide (FAD)-associated active site via a “shuttle disulfide” cysteine pair in Ero1 to protein disulfide isomerase (PDI) and from there on to substrate proteins [5], [6]. Mechanistically, all of these disulfide transfer reactions occur via interchain mixed-disulfide intermediates.

The synthesis of disulfide bonds in the ER, the compartment where secretory and membrane proteins are formed and folded, is essential. Not only reducing but also oxidizing disturbances, which compromise native disulfide-bond formation in the ER, result in locally hampered protein homeostasis—a state referred to as ER stress [9]. Increased Ero1 activity is a source of ER hyperoxidation and stress [10], [11], which is aggravated in the absence of the H2O2-detoxifying ER peroxidase GPx8 [12]. Accordingly, the catalytic rate of Ero1 enzymes requires tight negative feedback regulation to prevent Ero1-dependent toxicity [13].

In their inactive state, the “shuttle disulfide” cysteines (Cys94 and Cys99 in Ero1α or Cys90 and Cys95 in Ero1β) are engaged in intramolecular regulatory disulfides (Cys94–Cys131 and Cys99–Cys104 in Ero1α or Cys90–Cys130 and Cys95–Cys100 in Ero1β) [10], [11], [14], [15], [16]. However, although the inhibitory mechanism involving these disulfide bonds in mammalian Ero1 and their regulation by PDI family members is understood [17], [18], [19], it is surprising how well cells tolerate the overexpression of hyperactive Ero1 mutants lacking these disulfide bonds [10], [11], [20]. Furthermore, controversy exists as to how O2 reaches the active center in Ero1 and how H2O2 can be released again [5], [6].

Here, we report the existence of an additional regulated disulfide bond in mammalian Ero1, which is located at the distal side of the molecule relative to cofactor and "shuttle disulfide". When this disulfide is unlocked by reduced PDI, conformational rearrangements facilitate a diffusion pathway, through which O2 can reach the cofactor. Ero1 devoid of all regulatory disulfides is constitutively active, fails to associate with GPx8, and, consequently, produces cytotoxic levels of H2O2.

Section snippets

Cell culture and transient transfections

The culturing of HeLa cells [27] and FlipIn TRex293 cells for doxycycline (1 μg/ml, Sigma)-inducible expression of Ero1 variants [14] has been described. The following FlipIn TRex293 cell lines have been published previously: Ero1α [14], Ero1α-AA [10], Ero1α-AA:HyPerER [12], Ero1α-(AA+C85A/C391A) [10], Ero1β-C100A/C130A [27]. Corresponding cell lines with inducible expression of Ero1α-(AA+C94S), Ero1α-C208S/C241S, Ero1α-AASS, and Ero1β-AASS were generated equally. Ero1α-C208S/C241S:HyPerER and

Distinct Ero1α–PDI mixed disulfides reflect different activation states of Ero1α

Previous monitoring of Ero1α-dependent ER oxidation showed that treatment of cells with the disulfide reductant DTT activated not only wild-type Ero1α but also a hyperactive Ero1α mutant lacking all known regulatory disulfide bonds (Ero1α-C104A/C131A, in the following dubbed Ero1α-AA) [12]. Furthermore, DTT-mediated activation of Ero1α and Ero1β lowered the gel mobility of the Ero1–PDI mixed-disulfide complex [17]. Concentrating on the housekeeping isoform Ero1α, we first showed in

Discussion

The chemical basis for O2 reactivity of flavoenzymes is an actively investigated area in enzymology and cofactor biochemistry. In particular, how O2 can diffuse into the active sites of oxidases and monooxygenases has attracted ample interest [31]. Some of these flavoenzymes have well-defined channels predicted to funnel O2 to the FAD cofactor in a constitutive manner [32], [33], [34]. For many others, including Ero1, the molecular basis for O2 reactivity is a puzzling enigma [5], [6]. Here, by

Acknowledgments

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

References (57)

  • M.D. Shoulders et al.

    Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments

    Cell Rep

    (2013)
  • V.D. Nguyen et al.

    Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation

    J. Mol. Biol.

    (2011)
  • P. Chaiyen et al.

    The enigmatic reaction of flavins with oxygen

    Trends Biochem. Sci.

    (2012)
  • A. Alon et al.

    QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

    FEBS Lett.

    (2010)
  • D. Fass

    The Erv family of sulfhydryl oxidases

    Biochim. Biophys. Acta

    (2008)
  • S. Masui et al.

    Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI)

    J. Biol. Chem.

    (2011)
  • E. Gross et al.

    Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell

    Cell

    (2004)
  • E. Vitu et al.

    Oxidative activity of yeast Ero1p on protein disulfide isomerase and related oxidoreductases of the endoplasmic reticulum

    J. Biol. Chem.

    (2010)
  • C. Wang et al.

    Protein–protein docking with backbone flexibility

    J. Mol. Biol.

    (2007)
  • J.M. Villegas et al.

    FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

    Biochim. Biophys. Acta

    (2014)
  • K. Denoncin et al.

    Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead

    Antioxid. Redox Signaling

    (2013)
  • M. Fischer et al.

    The mitochondrial disulfide relay system: roles in oxidative protein folding and beyond

    Int. J. Cell Biol.

    (2013)
  • Y. Sato et al.

    Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals

    FEBS J.

    (2012)
  • K. Araki et al.

    Structure, mechanism, and evolution of Ero1 family enzymes

    Antioxid. Redox Signaling

    (2012)
  • T. Ramming et al.

    The physiological functions of mammalian endoplasmic oxidoreductin 1: on disulfides and more

    Antioxid. Redox Signaling

    (2012)
  • E. Gross et al.

    Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p

    Proc. Natl. Acad. Sci. USA

    (2006)
  • D. Eletto et al.

    Redox controls UPR to control redox

    J. Cell Sci.

    (2014)
  • H.G. Hansen et al.

    Biochemical evidence that regulation of Ero1beta activity in human cells does not involve the isoform-specific cysteine 262

    Biosci. Rep.

    (2014)
  • Cited by (0)

    1

    Current address: Berufsfachschule Gesundheit Baselland, 4142 Münchenstein, Switzerland.

    View full text