Oxidative and nitrative stress have been implicated in the molecular mechanisms underlying a variety of biological processes and disease states including cancer, aging, cardiovascular disease, neurological disorders, diabetes, and alcohol‐induced liver injury. One marker of nitrative stress is the formation of 3‐nitrotyrosine, or protein tyrosine nitration (PTN), which has been observed during inflammation and tissue injury; however, the role of PTN in the progression or possibly the pathogenesis of disease is still unclear. We show in a model of alcohol‐induced liver injury that an increase in PTN occurs in hepatocyte nuclei within the liver of wild‐type male C57BL/6J mice following chronic ethanol exposure (28 days). High‐resolution mass spectrometric analysis of isolated hepatic nuclei revealed several novel sites of tyrosine nitration on histone proteins. Histone nitration sites were validated by tandem mass spectrometry (MS/MS) analysis of representative synthetic nitropeptides equivalent in sequence to the respective nitrotyrosine sites identified in vivo. We further investigated the potential structural impact of the novel histone H3 Tyr41 (H3Y41) nitration site identified using molecular dynamics (MD) simulations. MD simulations of the nitrated and non‐nitrated forms of histone H3Y41 showed significant structural changes at the DNA interface upon H3Y41 nitration. The results from this study suggest that, in addition to other known post‐translational modifications that occur on histone proteins (e.g., acetylation and methylation), PTN could induce chromatin structural changes, possibly affecting gene transcription processes associated with the development of alcohol‐induced liver injury.

中文翻译：

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体内肝组蛋白硝化的位点特异性鉴定和验证：对酒精性肝损伤的影响

氧化应激和硝化应激与多种生物过程和疾病状态的分子机制有关，包括癌症、衰老、心血管疾病、神经系统疾病、糖尿病和酒精性肝损伤。硝化应激的标志之一是 3-硝基酪氨酸或蛋白质酪氨酸硝化 (PTN) 的形成，在炎症和组织损伤期间观察到这种现象；然而，PTN 在疾病进展或可能的发病机制中的作用仍不清楚。我们在酒精性肝损伤模型中发现，慢性乙醇暴露（28 天）后，野生型雄性 C57BL/6J 小鼠肝脏内的肝细胞核中 PTN 增加。对分离肝细胞核的高分辨率质谱分析揭示了组蛋白上酪氨酸硝化的几个新位点。通过对代表性合成硝基肽进行串联质谱 (MS/MS) 分析来验证组蛋白硝化位点，这些硝基肽的序列与体内鉴定的相应硝基酪氨酸位点相当。我们进一步研究了使用分子动力学 (MD) 模拟鉴定的新型组蛋白 H3 Tyr41 (H3Y41) 硝化位点的潜在结构影响。对组蛋白 H3Y41 硝化和非硝化形式的 MD 模拟显示，H3Y41 硝化后 DNA 界面发生显着的结构变化。这项研究的结果表明，除了组蛋白上发生的其他已知的翻译后修饰（例如乙酰化和甲基化）之外，PTN 还可以诱导染色质结构变化，可能影响与酒精诱导的酒精相关的基因转录过程。肝损伤。