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

Free Radic Biol Med. 2015 Jun;83:361-72. doi: 10.1016/j.freeradbiomed.2015.02.011. Epub 2015 Feb 17.

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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis*
  • Blotting, Western
  • Catalysis
  • Catalytic Domain
  • Cell Proliferation
  • Cells, Cultured
  • Disulfides / metabolism*
  • Endoplasmic Reticulum
  • Flavin-Adenine Dinucleotide / metabolism
  • Fluorescent Antibody Technique
  • HeLa Cells
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Molecular Chaperones / antagonists & inhibitors
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism
  • Oxidation-Reduction
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Oxygen / metabolism*
  • Protein Conformation
  • Protein Disulfide-Isomerases / chemistry
  • Protein Disulfide-Isomerases / genetics
  • Protein Disulfide-Isomerases / metabolism*
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Disulfides
  • ERP44 protein, human
  • Membrane Glycoproteins
  • Membrane Proteins
  • Molecular Chaperones
  • RNA, Messenger
  • RNA, Small Interfering
  • Flavin-Adenine Dinucleotide
  • Hydrogen Peroxide
  • ERO1A protein, human
  • Oxidoreductases
  • Protein Disulfide-Isomerases
  • Oxygen