Biochemical Mechanism of Eicosanoid Synthesizing Enzymes

项目摘要 饮食中ω-3和ω-6脂肪酸的摄入与心血管健康益处有关， 人类中心假设是ω-3和ω-6脂肪酸的心血管生理作用 部分通过环氧合酶（EPOX）途径合成类花生酸来介导。在此我们 对这些途径中的一些关键酶进行生物化学研究。CYP 2 J2是一种酶， EPOX途径在心血管系统的主动脉上皮中高度表达， 心肌细胞CYP 2 J2的主要作用是通过ω-3和ω-6脂肪酸环氧化为 环氧化物，发挥有效的抗炎，血管扩张和促血管生成的作用。CYP 2 J2也是 与药物的心脏毒性有关。此外，CYP 2 J2也是一种膜结合蛋白， 独特的生化机制，其特征很差，是目前的主要焦点。 提议我们的第一个目标是了解ω-3对CYP 2 J2表氧化酶活性的变构调节 和ω-6脂肪酸以及选定的心脏毒性药物（阿霉素、依巴斯汀和特非那定）。我们的第二 目的是检测CYP 2 J2对ω-3和ω-6脂肪酸衍生的内源性大麻素的代谢。是 与ω-6内源性大麻素类似，ω-3内源性大麻素是EPOX的底物， 产生新的生物活性环氧化物介质的酶。第三个目标是研究如何组成 影响CYP 2 J2活性。我们使用了几种新的方法，包括检测脂质 介质与质谱，创新的方法，如纳米盘溶解CYP 2 J2 并提供膜双层环境。我们还介绍了新的概念，脂质药物异向性 影响这些酶的产物形成的相互作用。这项工作的长期目标是 是了解从膳食脂肪酸形成类花生酸的相互作用。

PROJECT SUMMARY Dietary consumption of ω-3 and ω-6 fatty acids have been linked to cardiovascular health benefits in humans. The central hypothesis is that the cardiovascular physiological effects of ω-3 and ω-6 fatty acids are partly mediated by the synthesis of eicosanoids via the epoxygenase (EPOX) pathway. Herein we perform biochemical studies of some of the key enzymes in these pathways. CYP2J2 is an enzyme in the EPOX pathway that is highly expressed in the cardiovascular system in the aortic epithelium and cardiomyocytes. CYP2J2's primary effects are facilitated via epoxidation of ω-3 and ω-6 fatty acids into epoxides that exert potent anti- inflammatory, vasodilatory and pro-angiogenic effects. CYP2J2 is also implicated in cardiotoxicity of drugs. Additionally, CYP2J2 is also a membrane bound protein and exhibit unique biochemical mechanisms that are poorly characterized and are the primary focus of the current proposal. Our first goal is to understand allosteric modulation of CYP2J2 epoxygenase activity by ω-3 and ω-6 fatty acids and selected cardiotoxic drugs (doxorubicin, ebastine and terfenadine). Our second goal is to examine the metabolism of ω-3 and ω-6 fatty acid derived endocannabinoids by CYP2J2. It is predicted that similar to ω-6 endocannabinoids, the ω-3 endocannabinoids are substrates for the EPOX enzymes producing novel bioactive epoxide mediators. The third goal is to examine how the composition of membranes effect CYP2J2 activity. We use several novel approaches that includes detection of lipid mediators with mass spectrometry, innovative methodologies such as Nanodiscs to solubilize CYP2J2 and provide membrane bilayer environment. We also introduce novel concepts of lipid-drug heterotropic interactions influencing the formation of the products of these enzymes. The long-term goal of this work is to understand the interplay of the formation of the eicosanoids from dietary fatty acids.