S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography.
S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography.
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S-硫醇化拟态：通过谷胱甘肽亲和色谱法对蛋白质半胱氨酸的氧化还原状态进行定量和动力学分析。
DOI：
10.1016/j.abb.2005.10.013
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Srivenugopal,KalkunteS
中科院分区：
文献类型：
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作者：
Niture,SuryakantK;Velu,ChinavenmeniS;Bailey,NathanI;Srivenugopal,KalkunteS
S-谷胱甘肽化正在成为一种新型的调节和采用机制，通过该机制，谷胱甘肽（GSH 或 GSSG）缀合可以修饰氧化还原敏感蛋白中功能重要的反应性半胱氨酸。人们对细胞中这种修饰的产生和逆转的动态知之甚少。本研究描述了 GSH 和 GSSG 亲和基质定量结合含有反应性半胱氨酸并进行谷胱甘肽化的蛋白质的能力和适用性。我们表明，已知经过 S-硫醇化修饰的纯化蛋白质与这些基质结合，被二硫苏糖醇选择性洗脱，并在体外快速掺入生物素标记的 GSH 或 GSSG。用氧化剂（二酰胺、H2O2、叔丁基过氧化氢）处理过的肿瘤细胞提取物在 GSH-Sepharose 上进行色谱分析，结果显示许多蛋白质的特异性结合，其水平在处理后很快就会短暂增加（2 至 6 倍）。然而，当这些细胞在无药物/氧化剂的培养基中后孵育时，蛋白质结合在 3-12 小时内逐渐降低至控制水平，从而证明了半胱氨酸氧化还原状态在结合中的核心作用。 GSH-Sepharose 洗脱液的免疫印迹显示存在已知的（肌动蛋白、泛素激活酶 E1、NF-κB 和蛋白酶体）和推定的谷胱甘肽化靶标（p53、谷胱甘肽-S-转移酶 P1）。在氧化剂撤除后，许多这些蛋白质在失去与 GSH 基质的结合方面表现出独特的动力学，反映了它们从细胞环境中的半胱氨酸氧化还原变化中恢复的不同能力。此外，我们将蛋白酶体和泛素-E1 蛋白的 S-硫醇化敏感性动力学与 H2O2 处理的细胞中蛋白质泛素化水平的改变相关联。我们的研究揭示了谷胱甘肽基质在分析细胞蛋白中半胱氨酸氧化还原动力学方面迄今为止尚未充分利用的能力，并且可以轻松识别 S-硫醇化蛋白。
S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this modification in cells is poorly understood. This study describes the ability and applicability of GSH- and GSSG-affinity matrices to quantitatively bind proteins which harbor reactive cysteines and undergo glutathionylation. We showed that purified proteins, known to be modified by S-thiolation, bind to these matrices, are selectively eluted by dithiothreitol and rapidly incorporate biotin-labeled GSH or GSSG in vitro. Chromatography of extracts from tumor cells that had been treated with oxidants (diamide, H2O2, tert-butyl hydroperoxide) on GSH–Sepharose showed the specific binding of many proteins, whose levels increased transiently (2- to 6-fold) soon after treatments. However, when these cells were post-incubated in drug/oxidant-free media, protein binding decreased gradually to control levels over 3–12h, thereby demonstrating the central role of cysteine redox status in the binding. Immunoblotting of eluates from GSH–Sepharose showed the presence of known (actin, ubiquitin-activating enzyme E1, NF-κB, and proteasome) and putative (p53, glutathione-S-transferase P1) targets for glutathionation. After oxidant withdrawal, many of these proteins displayed unique kinetics in their loss of binding to GSH-matrix, reflecting their differential abilities to recover from cysteine redox changes in cellular milieu. Further, we correlated the kinetics of S-thiolation susceptibility of the proteasome and ubiquitin-E1 proteins with altered levels of protein ubiquitination in H2O2-treated cells. Our study reveals the hitherto underutilized ability of glutathione matrices for analyzing the kinetics of cysteine redox in cellular proteins and allows easy identification of S-thiolatable proteins.