Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems
Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems
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DOI：
10.1073/pnas.152168599
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发表时间：
2002-07-23
影响因子：
11.1
通讯作者：
Ghezzi, P
中科院分区：
文献类型：
--
作者：
Casagrande, S;Bonetto, V;Ghezzi, P
为了鉴定在人类T细胞母细胞中进行谷胱甘肽化（形成蛋白质-谷胱甘肽混合二硫化物）的蛋白质，我们用S-35放射性标记谷胱甘肽池，将细胞暴露于氧化剂二酰胺，并通过二维电泳分析细胞蛋白质。通过分子量、等电点和免疫印迹法鉴定其中一种谷胱甘肽化蛋白为硫氧还蛋白（Trx）。重组人Trx与谷胱甘肽二硫化物或S-亚硝基谷胱甘肽的温育导致谷胱甘肽化Trx的形成，通过基质辅助激光解吸电离飞行时间质谱法鉴定。谷胱甘肽化位点被鉴定为Cys-72。rhTrx的谷胱甘肽化在NADPH和Trx还原酶存在下消除了其作为胰岛素二硫还原酶的酶活性。然而，活动，恢复与S形动力学，表明一个过程中的自激活，由于能力的Trx去谷胱甘肽本身。这些数据表明，细胞内谷胱甘肽/谷胱甘肽二硫化物的比例，细胞的氧化还原状态的指标，可以调节Trx功能可逆地通过硫醇-二硫化物交换反应。
To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with S-35, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.