Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation.
Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation.
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DOI：
10.1006/abbi.1998.0859
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发表时间：
1998-10
影响因子：
3.9
通讯作者：
C. M. Padgett;A. Whorton
中科院分区：
文献类型：
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作者：
C. M. Padgett;A. Whorton
亚硝基硫醇是一氧化氮衍生物质（NOx）与硫醇反应的产物，参与细胞信号传导和细胞毒性事件。最近已证明，谷氨酸调节亚硝基硫醇介导的信号转导和保护免受NOx介导的细胞毒性。我们已经研究了蛋白S-硫醇化/去硫醇化作为谷胱甘肽调节亚硝基硫醇信号传导和毒性的潜在机制的作用。我们的数据表明，外源性来源的氮氧化物降低游离蛋白巯基和总谷胱甘肽水平的内皮细胞。谷胱甘肽水平的下降不能占形成S-亚硝基谷胱甘肽（GSNO），因为硼氢化物处理的非蛋白质部分的细胞提取物没有恢复谷胱甘肽水平，而硼氢化物处理的含蛋白质的细胞提取物导致谷胱甘肽水平的恢复。NOx介导的谷胱甘肽和蛋白质巯基含量的降低与蛋白质混合二硫键形成的增加相关，如通过将[35S]谷胱甘肽掺入细胞蛋白质所测量的。[35S]谷胱甘肽通过共价二硫键结合到蛋白质中，因为二硫苏糖醇从细胞蛋白质中去除了放射性标记。外源性氮氧化物源的撤回导致游离蛋白质巯基和细胞谷胱甘肽水平的恢复，这与蛋白质的去巯基化相关。去巯基化需要谷胱甘肽氧化还原系统的作用，因为谷胱甘肽还原酶抑制剂1，3-二（2-氯乙基）-1-亚硝基脲阻断谷胱甘肽水平的恢复和蛋白质的去巯基化。这些结果表明，细胞暴露于NOx不会导致GSNO的积累，而是刺激蛋白S-硫醇化，这是一种可能对亚硝基硫醇信号传导和毒性具有重要影响的机制。
Nitrosothiols, the product of the reaction of nitric oxide-derived species (NOx) with thiols, participate in both cell signaling and cytotoxic events. Glutathione has recently been shown to modulate nitrosothiol-mediated signal transduction and to protect against NOx-mediated cytotoxicity. We have investigated the role of protein S-thiolation/dethiolation as a potential mechanism by which glutathione regulates nitrosothiol signaling and toxicity. Our data show that exogenous sources of NOx decreased both free protein thiol and total glutathione levels in endothelial cells. The decrease in glutathione levels could not be accounted for by formation of S-nitrosoglutathione (GSNO) since borohydride treatment of the nonprotein fraction of cell extracts did not restore glutathione levels, whereas borohydride treatment of protein-containing cell extracts led to recovery of glutathione levels. The NOx-mediated decrease in glutathione and protein thiol content was correlated with an increase in protein mixed disulfide formation, as measured by the incorporation of [35S]glutathione into cellular proteins. [35S]glutathione was incorporated into proteins via a covalent disulfide bond since dithiothreitol removed the radiolabel from cellular proteins. The withdrawal of the exogenous NOx source led to recovery of free protein thiol and cellular glutathione levels, which correlated with the dethiolation of proteins. Dethiolation required the action of the glutathione redox system since 1, 3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of glutathione reductase, blocked both the recovery of glutathione levels and the dethiolation of proteins. These results suggest that exposure of cells to NOx does not lead to accumulation of GSNO but rather stimulates protein S-thiolation, a mechanism which may have important implications with respect to nitrosothiol signaling and toxicity.