Department of Medicine and University of California at Los Angeles, Los Angeles, CA 90095, USA.
J Lipid Res. 2012 Jul;53(7):1304-15. doi: 10.1194/jlr.M025320. Epub 2012 May 1.
Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), referred to as OxPAPC, and an active component, 1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphatidylcholine (PEIPC), accumulate in atherosclerotic lesions and regulate over 1,000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog of OxPAPC, covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine whether binding regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited the binding of OxPNB to HAEC proteins. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPNB and PEIPE-NB, the analog of PEIPC, bound to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ₂). This binding was observed with recombinant protein and in cells overexpressing H-Ras. OxPAPC and PEIPC compete with OxPNB for binding to H-Ras. 15dPGJ₂ and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC, PEIPC, and 15dPGJ₂ in HAEC, suggesting that some effects attributed to 15dPGJ₂ may also be regulated by PEIPC because both molecules accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function.
1-棕榈酰基-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（PAPC）的氧化产物，称为 OxPAPC，以及一种活性成分，1-棕榈酰基-2-（5,6-环氧异前列烷 E₂）-sn-甘油-3-磷酸胆碱（PEIPC），在动脉粥样硬化病变中积累，并调节人主动脉内皮细胞（HAEC）中的超过 1000 个基因。我们之前证明，OxPAPC 的生物素化类似物 OxPNB 与 HAEC 中的许多蛋白质共价结合。这些研究的目的是深入了解结合机制，并确定结合是否调节活性。在全细胞中，N-乙酰半胱氨酸抑制 OxPAPC 的基因调节，并用 N-乙基马来酰亚胺阻断细胞半胱氨酸强烈抑制 OxPNB 与 HAEC 蛋白的结合。使用 MS，我们证明 OxPAPC 与半胱氨酸的结合大部分是由 PEIPC 介导的。我们还表明，OxPNB 和 PEIPC 的类似物 PEIPE-NB，与以前显示可调节对 15-脱氧-Δ12,14-前列腺素 J2（15dPGJ₂）反应的活性的半胱氨酸结合模型蛋白 H-Ras。这种结合在重组蛋白和过表达 H-Ras 的细胞中都观察到。OxPAPC 和 PEIPC 与 OxPNB 竞争与 H-Ras 结合。15dPGJ₂和 OxPAPC 在可比浓度下增加 H-Ras 活性。使用微阵列分析，我们证明了 OxPAPC、PEIPC 和 15dPGJ₂在 HAEC 中的基因调节有相当大的重叠，表明归因于 15dPGJ₂的一些影响也可能受 PEIPC 调节，因为这两种分子都在炎症部位积累。总体而言，我们提供了证据表明 OxPAPC-半胱氨酸相互作用在调节 HAEC 功能方面的重要性。
