TY - JOUR
T1 - Cysteines 208 and 241 in Ero1α are required for maximal catalytic turnover
AU - Ramming, Thomas
AU - Kanemura, Shingo
AU - Okumura, Masaki
AU - Inaba, Kenji
AU - Appenzeller-Herzog, Christian
N1 - Funding Information:
This work was supported by a PhD fellowship by the Boehringer Ingelheim Fonds (to TR), the Swiss National Science Foundation (Ambizione) , the University of Basel , and the Freiwillige Akademische Gesellschaft (all to CAH), CREST, JST (to K. I.), Grant-in-Aids for Scientific Research on Innovative Areas from MEXT (to K. I. and M. O.), Takeda Science Foundation (to K. I.) and Grant-in-Aids for JSPS Fellows (to S. K. and M. O.).
Publisher Copyright:
© 2015 The Authors.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys94, Cys99, Cys394, Cys397), Ero1α harbors four regulatory cysteines (Cys104, Cys131, Cys208, Cys241). These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys208/Cys241 does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys208/Cys241 pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys208-Cys241 disulfide and the inhibitory disulfide bonds involving Cys104 and Cys131, which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol-disulfide exchange at the Cys208/Cys241 pair stimulates the catalytic turnover under reducing conditions are discussed.
AB - Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys94, Cys99, Cys394, Cys397), Ero1α harbors four regulatory cysteines (Cys104, Cys131, Cys208, Cys241). These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys208/Cys241 does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys208/Cys241 pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys208-Cys241 disulfide and the inhibitory disulfide bonds involving Cys104 and Cys131, which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol-disulfide exchange at the Cys208/Cys241 pair stimulates the catalytic turnover under reducing conditions are discussed.
KW - Disulfide bond
KW - Endoplasmic reticulum
KW - Ero1α
KW - PDI
KW - Redox homeostasis
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U2 - 10.1016/j.redox.2015.11.004
DO - 10.1016/j.redox.2015.11.004
M3 - Article
C2 - 26609561
AN - SCOPUS:84947811868
VL - 7
SP - 14
EP - 20
JO - Redox Biology
JF - Redox Biology
SN - 2213-2317
ER -