Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation
Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation
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DOI：
10.1074/jbc.m805287200
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发表时间：
2008-12-12
影响因子：
4.8
通讯作者：
Meng, Tzu-Ching
中科院分区：
文献类型：
--
作者：
Chen, Yi-Yun;Chu, Hsing-Mao;Meng, Tzu-Ching
由细胞一氧化氮（NO）介导的蛋白S-亚硝基化在cGMP非依赖性NO信号传导中执行生物学功能中起主要作用。虽然S-亚硝基化与活性氧诱导的Cys氧化相似，但其分子机制和生物学后果尚不清楚。研究了蛋白酪氨酸磷酸酶1B（PTP 1B）S-亚硝基化的结构过程。我们处理PTP 1B与各种NO供体，包括S-亚硝基硫醇试剂和化合物释放NO自由基，产生位点特异性的半胱氨酸S-亚硝基化鉴定使用先进的质谱（MS）技术。定量质谱分析表明，活性位点Cys-215是易发生S-亚硝基化的主要残基。在2.6埃分辨率下的NO供体反应的PTP 1B的晶体结构显示，在Cys-215处的S-NO状态没有可辨别的不可逆氧化形式，而其他Cys残基保持在其游离巯基状态。我们进一步证明了Cys-215残基的S-亚硝基化保护PTP 1B免受随后的H2 O2诱导的不可逆氧化。通过用NO供体预处理细胞或通过激活异位表达的NO合酶来增加细胞NO水平抑制活性氧诱导的内源性PTP 1B的不可逆氧化。这些结果表明，S-亚硝基化可能会防止PTPs的永久失活所造成的氧化应激。
Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 angstrom resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H2O2-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.