Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells.
Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells.
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DOI：
10.1371/journal.pone.0009075
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发表时间：
2010-02-05
期刊：
影响因子：
3.7
通讯作者：
Ho SM
中科院分区：
文献类型：
--
作者：
Lam YW;Yuan Y;Isaac J;Babu CV;Meller J;Ho SM
虽然一氧化氮合酶（NOS）的过度表达已被发现与前列腺疾病有关，NOS相关的前列腺疾病的潜在机制仍不清楚。一种提出的机制是与细胞信号通路中的关键调节蛋白的S-亚硝基化有关，这是由于前列腺中NO水平升高所致。因此，我们的主要目标是确定在永生化的正常前列腺上皮细胞系，NPrEC的S-亚硝基化的目标。我们用亚硝基半胱氨酸处理NPrEC，并使用生物素开关技术，然后进行基于凝胶的分离和质谱蛋白质鉴定（使用LTQ-Orbitrap），以发现处理过的细胞中的S-亚硝基化（SNO）蛋白。与此同时，我们采用了肽下拉方法来定位由第一种技术鉴定的蛋白质SNO靶标上的S-亚硝基化位点。这种组合方法鉴定了116种SNO蛋白，并确定了其中82种蛋白的修饰位点。这些蛋白质中超过60%属于四个功能组：细胞结构/细胞运动/蛋白质运输，蛋白质折叠/蛋白质反应/蛋白质组装，mRNA剪接/加工/转录调控和代谢。蛋白质印迹分析验证了与疾病发展相关的靶点子集（增殖细胞核抗原、maspin、整合素β4、α-连环蛋白、核转运蛋白[输入蛋白] β1和延伸因子1A 1）。我们分析了SNO序列的一级和二级结构、溶剂可及性和三维结构背景。我们发现，大约80%的SNO网站，可以映射到解决结构被掩埋，其中大约一半的带电氨基酸在其三维邻域，和另一半居住在主要疏水口袋。我们在这里确定了116个潜在的SNO靶点，并在NPrEC中绘制了它们的推定SNO位点。阐明这种翻译后修饰如何改变这些蛋白质的功能，应阐明NO在前列腺病理学中的作用。据我们所知，这是第一份在前列腺上皮细胞中鉴定SNO靶点的报告。
Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC. We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin β4, α-catenin, karyopherin [importin] β1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets. We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells.
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影响因子：
4.8
作者：
Azad, Neelam;Vallyathan, Val;Rojanasakul, Yon
通讯作者：
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影响因子：
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作者：
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DOI：
10.1016/j.urolonc.2007.05.007
发表时间：
2007-09-01
影响因子：
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作者：
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作者：
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