Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.
Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.
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DOI：
10.1021/tx200157t
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发表时间：
2011-08-15
影响因子：
4.1
通讯作者：
Liebler DC
中科院分区：
文献类型：
--
作者：
Connor RE;Marnett LJ;Liebler DC
亲电试剂修饰蛋白质的分析是一个具有挑战性的问题。大多数报道的蛋白质亲电加合物都是通过体外反应或通过加合物片段的亲和力捕获来表征的，这使得全局分析成为可能，但不适合特定蛋白质的靶向研究。我们采用靶向分子探针方法研究了调节多种客户蛋白的分子伴侣热休克蛋白90 （Hsp90）的修饰。用生物素-格尔达霉素探针非共价亲和力捕获从人RKO结直肠癌细胞中分离出两种天然蛋白（Hsp90α和Hsp90β）。格尔达那霉素-生物素捕获比免疫沉淀提供更高的Hsp90纯度，并且可以通过液相色谱-串联质谱（LC-MS/MS）检测内源性磷酸化蛋白。我们应用这种方法在RKO细胞中绘制和量化4-羟基烯醛（HNE）在Hsp90上形成的加合物。LC-MS/MS分析发现Hsp90α的His450和His490具有158 Da修饰，对应于nabh4还原的HNE加合物。Hsp90β上也被内合了5个组氨酸残基：His171、His442、His458、His625和His632。采用基于LC-MS/ ms的无标记相对定量方法，在体外用Hsp90蛋白测定这些位点的内聚率，在hne处理的细胞中用Hsp90蛋白测定这些位点的内聚率。在体外和细胞处理过程中，Hsp90α和Hsp90β上的残基内收率为3.0 × 10-5 h-1至1.08±0.17 h-1。在Hsp90α的中间客户端结合区域，Hsp90α处理的细胞中，残基His450内聚速度最快，kobs为1.08±0.17 h-1。Hsp90β上的同源残基His442比n端残基His171内合更快，尽管这两个残基的预测pKa值非常相似。因此，Hsp90中间客户端结合域可能在hne介导的Hsp90客户端蛋白相互作用的破坏中发挥重要作用。结果说明了蛋白质选择性亲和捕获方法用于亲电加合物及其生物学效应的靶向分析的实用性。
The analysis of protein modification by electrophiles is a challenging problem. Most reported protein–electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed a targeted molecular probe approach to study modifications of the molecular chaperone heat shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl–geldanamycin probe isolated both isoforms of the native protein (Hsp90α and Hsp90β) from human RKO colorectal cancer cells. Geldanamycin–biotin capture afforded higher purity Hsp90 than did immunoprecipitation and enabled detection of endogenously phosphorylated protein by liquid chromatography–tandem mass spectrometry (LC-MS/MS). We applied this approach to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. LC-MS/MS analyses of tryptic digests by identified His450 and His490 of Hsp90α as having a 158 Da modification, corresponding to NaBH4-reduced HNE adducts. Five histidine residues were also adducted on Hsp90β: His171, His442, His458, His625, and His632. The rates of adduction at these sites were determined with Hsp90 protein in vitro and with Hsp90 in HNE-treated cells with a LC-MS/MS-based, label-free relative quantitation method. During in vitro and cell treatment with HNE, residues on Hsp90α and Hsp90β displayed adduction rates ranging from 3.0 × 10–5 h–1 to 1.08 ± 0.17 h–1. Within the middle client-binding domain of Hsp90α, residue His450 demonstrated the most rapid adduction with kobs of 1.08 ± 0.17 h–1 in HNE-treated cells. The homologous residue on Hsp90β, His442, was adducted more rapidly than the N-terminal residue, His171, despite very similar predicted pKa values of both residues. The Hsp90 middle client-binding domain thus may play a signicant role in HNE-mediated disruption of Hsp90–client protein interactions. The results illustrate the utility of a protein-selective affinity capture approach for targeted analysis of electrophile adducts and their biological effects.
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DOI：
10.1074/mcp.m800070-mcp200
发表时间：
2009-04
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Codreanu SG;Zhang B;Sobecki SM;Billheimer DD;Liebler DC
通讯作者：
Liebler DC
影响因子：
2.9
作者：
Patricelli, Matthew P.;Szardenings, A. Katrin;Kozarich, John W.
通讯作者：
Kozarich, John W.
影响因子：
4.4
作者：
Hansen, BT;Davey, SW;Liebler, DC
通讯作者：
Liebler, DC
影响因子：
4.1
作者：
Carbone, DL;Doorn, JA;Petersen, DR
通讯作者：
Petersen, DR
DOI：
10.1124/jpet.105.088088
发表时间：
2005-10-01
影响因子：
3.5
作者：
Carbone, DL;Doorn, JA;Petersen, DR
通讯作者：
Petersen, DR