Expression and Post-translational Modification of Cytochrome P450 4A11 Variants in Modulation of Blood Pressure Through Regulation of 20-Hydroxyeicosatetraenoic Acid (20-HETE) Production

摘要 高血压是一种复杂的疾病，影响40%的25岁以上的人。减少在血 压力与冠心病和中风的显著减少有关。我们漫长 长期目标是在细胞色素P450的背景下开发新的高血压治疗策略 系统细胞色素P450（CYP）4A 11是一种β-羟化酶，催化花生四烯酸的氧化 (AA)至20-羟基二十碳四烯酸（20-HETE）。20-HETE可以通过收缩 微血管或通过抑制肾脏中的盐重吸收来抗高血压。因此，20-HETE和 CYP 4A 11在血压调节中具有重要作用。在人类中，CYP 4A 11的一种遗传变体 （rs 1126742 T8590 C等位基因）编码具有降低的β-羟基化活性的蛋白质（CYP 4A 11-F434 S）， 与高血压和对某些利尿剂的抗高血压作用的抵抗有关。 这种明显的失调，连同F434 S变体中20-HETE活性的降低，表明 其他未知因素可能有助于CYP 4A 11的促高血压和抗高血压作用。中央 假设表达和/或翻译后修饰差异地影响 CYP 4A 11与20-HETE合成有关，并最终控制血压。CYP 4A 11调节 和/或酶活性将测试对20-HETE水平的影响和活性氧的影响 物种（ROS）。生物化学，分析和酶学工具的组合将用于进一步研究 阐明CYP 4A 11 Phe-434和Ser-434变体在底物选择、活性 和蛋白质水平。首先，化学选择性探针的使用将确定氧化还原状态在细胞中的作用。 CYP 4A 11的翻译后调节。接下来，研究CYP 4A 11变异蛋白 将在人微粒体中研究花生四烯酸的浓度和代谢活性， 转基因小鼠模型。这些实验旨在进一步阐明CYP 4A 11变异体在 高血压20-HETE是高血压的重要组成部分。基因变异改变了它的生产 引起高血压易感性和治疗反应的显著变化。了解 与CYP 4A 11表型变异相关的机制将有助于更深入地了解 不同个体的高血压，并重塑这些特定人群的血压治疗方式 还原

ABSTRACT Hypertension is a complex condition that affects 40% of persons over the age of 25 years. Reduction in blood pressure has been associated with a significant decrease in both coronary heart disease and stroke. Our long term goal is to develop novel therapeutic strategies for hypertension in the context of the cytochrome P450 system. Cytochrome P450 (CYP) 4A11 is an -hydroxylase that catalyzes the oxidation of arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE can be pro-hypertensive by constricting microvasculature or anti-hypertensive by inhibiting salt reabsorption in the kidney. Thus, 20-HETE and CYP4A11 have important roles in blood pressure regulation. In humans, a genetic variant of CYP4A11 (rs1126742 T8590C allele) encodes for a protein (CYP4A11-F434S) with reduced -hydroxylation activity that has been associated with high blood pressure and resistance to the anti-hypertensive effects of some diuretics. This apparent deregulation, in conjunction with decreased 20-HETE activity in the F434S variant, suggests that other unknown factors may contribute to the pro- and anti-hypertensive effects of CYP4A11. The central hypothesis is that expression and/or posttranslational modification differentially effects genetic variants of CYP4A11 with regard to 20-HETE synthesis and, ultimately, blood pressure control. CYP4A11 regulation and/or enzymatic activity will be tested for effects on 20-HETE levels and for influences by reactive oxygen species (ROS). A combination of biochemical, analytical, and enzymological tools will be used to further elucidate the differences between the CYP4A11 Phe-434 and Ser-434 variants in substrate selection, activity, and protein level. First, the use of chemoselective probes will determine the role of redox status in the posttranslational regulation in CYP4A11. Next, the relationship between CYP4A11 variant protein concentration and arachidonic acid metabolizing activity will be investigated in human microsomes and a transgenic mouse model. These experiments aim to further elucidate the role CYP4A11 variants play in hypertension. 20-HETE is an important component in hypertension. Genetic variations that alter its production cause significant changes in hypertension susceptibility and therapeutic response. Understanding the mechanisms related to the phenotypic variation in CYP4A11 will help to gain a more in-depth understanding of hypertension in different individuals and reshape how these specific persons are treated for blood pressure reduction.