S-glutathionylation uncouples eNOS and regulates its cellular and vascular function.
S-glutathionylation uncouples eNOS and regulates its cellular and vascular function.
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DOI：
10.1038/nature09599
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发表时间：
2010-12-23
期刊：
影响因子：
64.8
通讯作者：
Zweier, Jay L.
中科院分区：
文献类型：
--
作者：
Chen, Chun-An;Wang, Tse-Yao;Varadharaj, Saradhadevi;Reyes, Levy A.;Hemann, Craig;Talukder, M. A. Hassan;Chen, Yeong-Renn;Druhan, Lawrence J.;Zweier, Jay L.
内皮型一氧化氮合酶（eNOS）在调节血管功能中至关重要，可以产生一氧化氮（NO）和超氧化物（O2·−），这是细胞信号传导的关键介质。在Ca 2 +/钙调蛋白存在下，eNOS通过电子从NADPH通过含黄素的还原酶结构域转移到结合在加氧酶结构域的血红素处的氧，从L-精氨酸（L-Arg）产生NO（内皮源性舒张因子），所述加氧酶结构域还含有四氢生物蝶呤（BH 4）和L-Arg的结合位点。在没有BH 4的情况下，NO的合成被废除，而生成O2·−。虽然NOS功能障碍发生在氧化还原应激疾病中，但BH 4补充仅部分恢复NOS活性和NOS依赖性血管舒张。这表明，有一个尚未确定的氧化还原调节机制控制NOS功能。蛋白质硫醇可以进行S-谷胱甘肽化，这是一种参与细胞信号传导和适应的可逆蛋白质修饰。在氧化应激下，S-谷胱甘肽化通过与氧化型谷胱甘肽的巯基-二硫化物交换或氧化剂诱导的蛋白质硫酰基自由基与还原型谷胱甘肽的反应发生。半胱氨酸残基是维持eNOS功能的关键，因此我们推测氧化应激可以通过S-谷胱甘肽化改变eNOS活性。在这里，我们表明，eNOS的S-谷胱甘肽化可逆地降低NOS活性，增加主要来自还原酶的O2·−生成，其中两个高度保守的半胱氨酸残基被确定为S-谷胱甘肽化位点，并发现对eNOS功能的氧化还原调节至关重要。我们发现内皮细胞中eNOS S-谷胱甘肽化，伴随着NO的损失和O2·−生成的增加，与内皮依赖性血管舒张功能受损相关。在高血压血管中，eNOS S-谷胱甘肽化随着内皮依赖性血管舒张受损而增加，该内皮依赖性血管舒张通过巯基特异性还原剂恢复，巯基特异性还原剂逆转该S-谷胱甘肽化。因此，eNOS的S-谷胱甘肽化是提供细胞信号传导、内皮功能和血管张力的氧化还原调节的关键开关。
Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O2•−), which are key mediators of cellular signalling. In the presence of Ca2+/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from L-arginine (L-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH4) and L-Arg. In the absence of BH4, NO synthesis is abrogated and instead O2•− is generated. While NOS dysfunction occurs in diseases with redox stress, BH4 repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol–disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O2•− generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O2•− generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.
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