Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

中性粒细胞是包括缺血性心脏病、动脉粥样硬化和败血症等疾病中损伤和修复的重要细胞介质。中性粒细胞释放的髓过氧化物酶衍生（MPO）氧化剂是疾病中内皮损伤的潜在介质。MPO衍生的次氯酸（HOCl）攻击缩醛磷脂以释放2 - 氯脂肪酸醛（2 - ClFALD）。2 - ClFALD及其氧化产物2 - 氯脂肪酸（2 - ClFA）均为亲电脂质，两者可能通过多种机制与蛋白质发生反应。在本研究中，我们在非还原条件下（例如，不稳定希夫碱键的情况下）专门研究2 - ClFALD对蛋白质的修饰，这可能反映了亲电氯化碳的亲核靶向作用。在多个细胞系中，将2 - 氯十六醛（2 - ClHDA）的ω - 炔烃类似物2 - ClHDyA对蛋白质的修饰与2 - 氯十六酸（2 - ClHA）的ω - 炔烃类似物2 - ClHyA对蛋白质的修饰进行了比较，结果表明与2 - ClFA相比，2 - ClFALD优先修饰蛋白质。通过鸟枪法蛋白质组学和过度表达分析对EA.hy926细胞和人肺微血管内皮细胞中2 - ClHDyA修饰的蛋白质进行分析，结果包括黏附连接、细胞黏附分子结合和细胞底物连接富集类别。这些组中的蛋白质可能在先前描述的2 - ClFALD引发的内皮屏障功能障碍中起作用。