Integrated redox sensor and effector functions for tetrahydrobiopterin- and glutathionylation-dependent endothelial nitric-oxide synthase uncoupling.
Integrated redox sensor and effector functions for tetrahydrobiopterin- and glutathionylation-dependent endothelial nitric-oxide synthase uncoupling.
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DOI：
10.1074/jbc.m112.415992
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发表时间：
2013-01-04
期刊：
影响因子：
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通讯作者：
Channon KM
中科院分区：
文献类型：
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作者：
Crabtree MJ;Brixey R;Batchelor H;Hale AB;Channon KM
背景：四氢生物蝶呤（tetrahydrobiopterin，BH 4）和S-谷胱甘肽化都是eNOS活性和偶联的重要调节因子。结果如下：S-谷胱甘肽化和BH 4缺乏通过不同的机制诱导eNOS解偶联，但通过BH 4氧化和细胞GSH：GSSG比率的变化相互调节。结论：BH 4依赖性和S-谷胱甘肽化诱导的eNOS解偶联在机制上是独立的，但在功能上是相关的。重要性：BH 4和S-谷胱甘肽化使eNOS成为一个整合的氧化还原信号“枢纽”。内皮型一氧化氮合酶（eNOS）是血管内稳态的重要调节因子，通过产生依赖于辅因子四氢生物蝶呤（BH 4）的NO。当BH 4的可用性是有限的，eNOS成为“解偶联”，导致超氧化物的产生，而不是NO。最近的证据表明，eNOS解偶联也可以诱导S-谷胱甘肽化，虽然BH 4和S-谷胱甘肽化之间的功能关系仍然未知。为了解决BH 4在S-谷胱甘肽化诱导的eNOS解偶联中的可能作用，我们表达了WT或突变型eNOS，使其在具有Tet-regulated表达的人GTP环化水解酶I的细胞中对S-谷胱甘肽化具有抗性，以调节细胞内BH 4的可用性。我们发现，通过暴露于1，3-双（2-氯乙基）-1-亚硝基脲（BCNU）或谷胱甘肽还原酶特异性siRNA，eNOS的S-谷胱甘肽化导致NO产生减少和eNOS衍生的超氧化物产生升高，沿着BH 4水平和BH 4：7，8-二氢生物蝶呤比率的降低。在BH 4缺乏诱导的eNOS解偶联中，BCNU暴露进一步加剧了超氧化物的产生，BH 4氧化和eNOS活性。C908 S突变后，BCNU诱导的eNOS解偶联和BH 4氧化被废除，而BH 4缺乏诱导的解偶联被保留。此外，BH 4缺乏单独足以降低细胞内GSH：GSSG比率并引起eNOS S-谷胱甘肽化。这些数据提供了第一个证据表明，BH 4缺乏和S-谷胱甘肽诱导的eNOS解偶联机制，虽然机制不同，但功能相关。我们建议，解偶联的eNOS的S-谷胱甘肽-或BH 4-依赖的机制，使eNOS作为一个综合的氧化还原“枢纽”连接上游的BH 4和谷胱甘肽的氧化还原敏感性的影响与氧化还原依赖的目标和途径，位于eNOS的下游。
Background: Both tetrahydrobiopterin (BH4) and S-glutathionylation are important regulators of eNOS activity and coupling. Results: S-Glutathionylation and BH4 deficiency induce eNOS uncoupling through distinct mechanisms but are mutually regulated by changes in BH4 oxidation and cellular GSH:GSSG ratio. Conclusion: BH4-dependent and S-glutathionylation-induced eNOS uncoupling are mechanistically independent but functionally linked. Significance: BH4 and S-glutathionylation exemplify eNOS as an integrated redox signaling “hub.” Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes “uncoupled,” resulting in superoxide production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, although the functional relationships between BH4 and S-glutathionylation remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S-glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability. We reveal that S-glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4 oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S-glutathionylation. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We propose that uncoupling of eNOS by S-glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox “hub” linking upstream redox-sensitive effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.