Subcellular targeting and differential S-nitrosylation of endothelial nitric-oxide synthase
Subcellular targeting and differential S-nitrosylation of endothelial nitric-oxide synthase
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DOI：
10.1074/jbc.m510421200
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发表时间：
2006-01-06
影响因子：
4.8
通讯作者：
Michel, T
中科院分区：
文献类型：
--
作者：
Erwin, PA;Mitchell, DA;Michel, T
内皮一氧化氮合酶（eNOS）经历了一个复杂的翻译后修饰模式，调节其活性。我们最近报道eNOS在内皮细胞中构成s -亚硝基化，并且激动剂在酶激活的同时促进eNOS去硝基化(Erwin， P. A., Lin， A. J., Golan， D. E., and Michel， T. (2005)， J. Biol。化学，19888 - 19894)。在本研究中，我们用质谱法证实了eNOS的四硫酸锌半胱氨酸是s -亚硝基化的。eNOS靶向质膜是s -亚硝基化酶的必要条件，我们报道了细胞间的易位是动态eNOS s -亚硝基化的必要条件。我们用编码野生型eNOS的cDNA转染细胞，这是膜靶向的，或者用酰基化缺陷突变型eNOS （Myr(-)）转染细胞，这只在细胞质中表达。虽然野生型eNOS是强有力的s -亚硝基化，但我们发现Myr(-) eNOS突变体的s -亚硝基化几乎被消除。当我们用Myr(-) eNOS连接到cd8 -跨膜结构域（CD8-Myr(-)）的融合蛋白转染细胞时，我们发现CD8-Myr(-) eNOS不经历动态亚细胞易位，相对于野生型eNOS是高亚硝基化的。此外，我们发现转染了野生型或CD8-Myr(-) eNOS的内皮细胞用eNOS激动剂刺激时，只有野生型eNOS发生脱硝基化；CD8-Myr(-) enoss -亚硝基化不变。这些发现表明亚细胞靶向是eNOS s -亚硝基化的关键决定因素。最后，我们发现eNOS s -亚硝基化可以在小鼠完整动脉制剂中检测到，并且eNOS s -亚硝基化在完整血管中是一个动态的激动剂调节过程。这些研究表明，受体调控的enoss -亚硝基化可能是血管壁no依赖性信号传导的重要决定因素。
Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005), J. Biol. Chem. 280, 19888 - 19894). In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wildtype eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr(-)), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr(-) eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr(-) eNOS is ligated to the CD8-transmembrane domain (CD8-Myr(-)), we found that CD8-Myr(-) eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr(-) eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr(-) eNOSS-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOSS-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall.