title: In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms.
abstract: Thiol-disulfide oxidoreductases of the human protein disulfide isomerase (PDI) family promote protein folding in the endoplasmic reticulum (ER), while also assisting the retrotranslocation of toxins and misfolded ER proteins to the cytosol. The redox activity of PDI-like proteins is determined by the redox state of active-site cysteines found in a Cys-Xaa-Xaa-Cys motif. Progress in understanding redox regulation of the mammalian enzymes is currently hampered by the lack of reliable methods to determine quantitatively their redox state in living cells. We developed such a method based on the alkylation of cysteines by methoxy polyethylene glycol 5000 maleimide. With this method, we showed for the first time that in vivo PDI is present in two semi-oxidized forms in which either the first active site (in the a domain) or the second active site (in the a' domain) is oxidized. We report a steady-state redox distribution of endogenous PDI in HEK-293 cells of 50 +/- 5% fully reduced, 18 +/- 2% a-oxidized/a' -reduced, 15 +/- 2% a-reduced/a' -oxidized, and 16 +/- 4% fully oxidized. These results suggest that neither of the two domains in human PDI exclusively catalyzes substrate oxidation or reduction in vivo.
authors.affiliation: Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), ETH-Hoenggerberg, Zurich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Appenzeller-Herzog
authors.forename: Lars
authors.initials: L
authors.lastname: Ellgaard
mesh_terms: D000478:Alkylation; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047468:Immunoprecipitation; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D017931:DNA Primers; D019704:Protein Disulfide-Isomerases