In vivo reduction-oxidation state of protein disulfide isomerase:: The two active sites independently occur in the reduced and oxidized forms
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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DOI：
10.1089/ars.2007.1837
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发表时间：
2008-01-01
影响因子：
6.6
通讯作者：
Ellgaard, Lars
中科院分区：
文献类型：
--
作者：
Appenzeller-Herzog, Christian;Ellgaard, Lars
人类蛋白质二硫键异构酶 (PDI) 家族的硫醇二硫键氧化还原酶促进内质网 (ER) 中的蛋白质折叠，同时还协助毒素和错误折叠的 ER 蛋白质逆向易位至胞质溶胶。 PDI 样蛋白的氧化还原活性由 Cys-Xaa-Xaa-Cys 基序中活性位点半胱氨酸的氧化还原状态决定。目前，由于缺乏可靠的方法来定量确定活细胞中的氧化还原状态，理解哺乳动物酶的氧化还原调节的进展受到阻碍。我们开发了一种基于甲氧基聚乙二醇 5000 马来酰亚胺对半胱氨酸进行烷基化的方法。通过这种方法，我们首次证明体内 PDI 以两种半氧化形式存在，其中第一个活性位点（在 a 结构域中）或第二个活性位点（在 a' 结构域中）被氧化。我们报告了内源性的稳态氧化还原分布； HEK-293 细胞中的 PDI 为 50 +/- 5% 完全还原、18 +/- 2% a-氧化/a'-还原、15 +/- 2% a-还原/a'-氧化和 16 +/- 4% 完全氧化。这些结果表明，人 PDI 中的两个结构域都不专门催化体内底物氧化或还原。
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-Iike 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 semioxidized 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.