In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms
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In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms. / Appenzeller-Herzog, Christian; Ellgaard, Lars.
I: Antioxidants & Redox Signaling, Bind 10, Nr. 1, 2008, s. 55-64.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms
AU - Appenzeller-Herzog, Christian
AU - Ellgaard, Lars
N1 - Keywords: Alkylation; Animals; Base Sequence; Binding Sites; Blotting, Western; Catalysis; Cell Line; Cercopithecus aethiops; DNA Primers; Electrophoresis, Polyacrylamide Gel; Humans; Immunoprecipitation; Oxidation-Reduction; Protein Disulfide-Isomerase; Vero Cells
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
U2 - 10.1089/ars.2007.1837
DO - 10.1089/ars.2007.1837
M3 - Journal article
C2 - 17939758
VL - 10
SP - 55
EP - 64
JO - Antioxidants and Redox Signaling
JF - Antioxidants and Redox Signaling
SN - 1523-0864
IS - 1
ER -
ID: 9021102