Padgett C M, Whorton A R
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
Arch Biochem Biophys. 1998 Oct 15;358(2):232-42. doi: 10.1006/abbi.1998.0859.
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.
亚硝基硫醇是一氧化氮衍生物种（NOx）与硫醇反应的产物，参与细胞信号传导和细胞毒性事件。最近研究表明，谷胱甘肽可调节亚硝基硫醇介导的信号转导，并预防NOx介导的细胞毒性。我们研究了蛋白质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所起的作用。我们的数据表明，外源性NOx降低了内皮细胞中的游离蛋白质硫醇和总谷胱甘肽水平。谷胱甘肽水平的降低不能用S-亚硝基谷胱甘肽（GSNO）的形成来解释，因为用硼氢化钠处理细胞提取物的非蛋白质部分并不能恢复谷胱甘肽水平，而用硼氢化钠处理含蛋白质的细胞提取物则可使谷胱甘肽水平恢复。通过将[35S]谷胱甘肽掺入细胞蛋白质中测定，NOx介导的谷胱甘肽和蛋白质硫醇含量的降低与蛋白质混合二硫键形成的增加相关。[35S]谷胱甘肽通过共价二硫键掺入蛋白质中，因为二硫苏糖醇可去除细胞蛋白质中的放射性标记。去除外源性NOx来源后，游离蛋白质硫醇和细胞谷胱甘肽水平恢复，这与蛋白质的去硫醇化相关。去硫醇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-双（2-氯乙基）-1-亚硝基脲可阻断谷胱甘肽水平的恢复和蛋白质的去硫醇化。这些结果表明，细胞暴露于NOx不会导致GSNO积累，而是刺激蛋白质S-硫醇化，这一机制可能对亚硝基硫醇信号传导和毒性具有重要意义。
