Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.
Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.
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DOI：
10.1074/mcp.m800070-mcp200
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发表时间：
2009-04
期刊：
影响因子：
--
通讯作者：
Liebler DC
中科院分区：
文献类型：
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作者：
Codreanu SG;Zhang B;Sobecki SM;Billheimer DD;Liebler DC
脂质过氧化产生多种亲电子试剂，这些亲电子试剂被认为有助于涉及氧化应激的疾病的分子发病机制，但人们对脂质亲电子试剂引起的蛋白质损伤的范围知之甚少。我们在用 50 或 100 μm HNE 处理的 RKO 细胞中鉴定了原型脂质亲电子试剂 4-羟基-2-壬烯醛 (HNE) 的蛋白质靶点。通过与生物胺氨基己酸酰肼反应将HNE Michael加合物生物素化，用链霉亲和素捕获，捕获的蛋白质通过一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，用胰蛋白酶消化，并通过液相色谱-串联质谱鉴定。在已识别的 1500 多种蛋白质中，有 417 种蛋白质随着 HNE 暴露浓度的增加而表现出统计学上显着的内收增加。我们通过使用抗生物素抗体进行特异性捕获并通过高分辨率液相色谱-串联质谱法进行分析，进一步鉴定了 18 种生物素酰肼修饰的 HNE 加合肽。使用链霉亲和素捕获和免疫印迹方法对已识别的 HNE 靶标的子集进行了验证，该方法能够在 HNE 暴露量低至 1 μm 时检测加合物。蛋白质相互作用网络分析表明，氧化应激中的几个子系统受到内源性亲电子试剂的影响，包括参与蛋白质折叠和降解的 26S 蛋白酶体和伴侣蛋白 TCP-1 (CCT) 系统，以及 COP9 信号体、翻译起始复合物和大型核糖核蛋白网络。蛋白质脂质亲电子加合物的整体分析为涉及氧化应激的疾病机制提供了系统水平的视角。
Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 μm HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 μm. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.