A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells.
Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.
Appenzeller-Herzog
Christian
C
Department of Biology, University of Copenhagen, Copenhagen Ø, Denmark.
Riemer
Jan
J
Christensen
Brian
B
Sørensen
Esben S
ES
Ellgaard
Lars
L
D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism
D004220:Disulfides; D007527:Isoenzymes; D008562:Membrane Glycoproteins; C405327:ERO1A protein, human; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine