Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress
Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress
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DOI：
10.1074/jbc.m609684200
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发表时间：
2007-05-11
影响因子：
4.8
通讯作者：
Stamler, Jonathan S.
中科院分区：
文献类型：
--
作者：
Forrester, Michael T.;Foster, Matthew W.;Stamler, Jonathan S.
蛋白质S-亚硝基化已成为一氧化氮发挥生物学效应的主要机制。在研究蛋白质S-亚硝基化的方法中，生物素开关技术（BST）由于其可以容易地检测生物样品中的单个S-亚硝基化（SNO）蛋白而迅速流行。通过BST鉴定SNO位点依赖于抗坏血酸盐从S-亚硝基硫醇产生硫醇的能力，但不是从S-氧化硫醇（例如二硫化物、次磺酸）产生硫醇的能力。然而，这种反应的特异性最近受到了挑战，促使一些人声称BST可能产生假阳性结果，并对BST在氧化条件下的应用提出了担忧。在这里，我们进行了比较分析的BST使用差异S-氧化和S-亚硝基化形式的蛋白酪氨酸磷酸酶1B，以及完整的和裂解的人胚肾293细胞处理的S-氧化和S-亚硝基化剂，并验证该测定是高度特异性的SNO。引人注目的是，从实验室窗户的间接阳光下暴露的样品导致人为的抗坏血酸依赖的信号，这可能是由半氢抗坏血酸自由基促进;从阳光的保护消除了伪影。相比之下，在BST之前将SNO蛋白暴露于强紫外光源（SNO光解）提供了测定特异性的独立验证。通过将BST与光解结合，我们已经表明抗癌药物诱导的氧化应激促进主要凋亡效应物甘油醛-3-磷酸脱氢酶的S-亚硝基化。总的来说，这些实验表明，SNO依赖性信号通路可以通过氧化条件进行调节，并表明S-亚硝基化在药物作用中的潜在作用。
Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.