CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

心脏细胞色素 P450 花生四烯酸环氧化酶途径

• 批准号: 6106635
• 负责人: Darryl C Zeldin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106635
• 关键词: arachidonate; clinical research; cytochrome P450; eicosanoid metabolism; enzyme activity; epoxides; gene expression; genetic promoter element; genetically modified animals; heart cell; heart function; hemoprotein metabolism; human subject; laboratory mouse; myocardial ischemia /hypoxia; oxygenases; reperfusion

The human CYP2J2 cDNA has been cloned, expressed in Sf9 insect cells, and extensively characterized with respect to its tissue distribution, cellular localization and function. This enzyme appears to be the major human P450 expressed in heart, where it is highly localized to cardiac myocytes and endothelial cells, and highly active in the metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) and 19-hydroxyeicosatetraenoic acid (19-HETE). Interestingly, one of the EETs (11,12-EET) improves cardiac contractility following prolonged global ischemia, causes hyperpolarization of the resting membrane potential and shortening of the cardiac action potential, and markedly inhibits cardiac L-type Ca++ channel activity. The human CYP2J2 gene has been cloned, characterized with respect to its intron/exon organization, and mapped to Chromosome 1p31.3-31.2 in close proximity to the CYP4A and CYP4B loci. The CYP2J2 gene appears to be regulated, in part, by alternative splicing at the exon 1/intron 1 junction. Two alternative splice variants (CYP2J2-H2 and CYP2J2-H5) have been cloned and shown to be expressed in multiple tissues including the heart by RT-PCR and protein immunoblotting. We have also identified a polymorphic variant of the CYP2J2 gene that results in a T to A substitution at position 143 within the C-helix in close proximity to one of the putative P450 substrate recognition sites. These and other CYP2J2 variants will be identified and functionally characterized as part of the NIEHS Environmental Genome Project. Efforts are currently underway to: (a) construct endothelial and vascular smooth muscle cell lines that stably overexpress CYP2J2 to examine the effects of CYP2J2 products on Ca++ signaling; (b) co-express human CYP2J2 and green fluorescent protein in cultured neonatal rat heart myocytes to examine the role of this enzyme and its products on cardiac myocyte function under basal conditions and during metabolic stress; and (c) construct transgenic mice that overexpress CYP2J2 in heart muscle to examine the effects of CYP2J2-derived eicosanoids on cardiac function in vivo. In related studies, we have identified five novel mouse Cyp2j genes, at least two of which (Cyp2j6 and Cyp2j8) appear to be expressed in mouse heart by RT-PCR analysis. We have cloned the corresponding cDNAs, and are in the process of expressing the recombinant proteins in Sf9 insect cells to examine their AA metabolic activity. Preliminary studies suggest that the CYP2J6 heme-thiolate protein is unstable. This has precluded enzymatic studies and analysis of the distribution of CYP2J6 protein using a peptide-based antibody. We are currently investigating ways to stabilize the recombinant CYP2J6 so that these studies will be possible.

人类 CYP2J2 cDNA 已被克隆， 在 Sf9 昆虫细胞中表达，并广泛表征为 关于其组织分布、细胞定位和功能。 该酶似乎是人类 P450 中表达的主要酶 心脏，高度定位于心肌细胞和 内皮细胞，花生四烯酸的代谢高度活跃 酸（AA）到环氧二十碳三烯酸（EET）和 19-羟基二十碳四烯酸（19-HETE）。有趣的是，其中之一 EET (11,12-EET) 改善心肌收缩力 长时间的全身缺血，导致静息状态的超极化 膜电位和心脏动作电位的缩短， 并显着抑制心脏 L 型 Ca++ 通道活性。这 人类 CYP2J2 基因已被克隆，其特征在于 其内含子/外显子组织，并映射到染色体 1p31.3-31.2 非常接近 CYP4A 和 CYP4B 位点。这 CYP2J2 基因似乎部分受到替代性调控 剪接于外显子 1/内含子 1 连接处。两种替代拼接 变体（CYP2J2-H2 和 CYP2J2-H5）已被克隆并 显示在包括心脏在内的多种组织中表达 RT-PCR 和蛋白质免疫印迹。我们还确定了一个 CYP2J2 基因的多态性变体，导致 T 到 A 在 C 螺旋内的 143 位取代，紧邻 假定的 P450 底物识别位点之一。这些和 其他 CYP2J2 变体将被识别并发挥功能 作为 NIEHS 环境基因组计划的一部分。 目前正在努力：（a）构建内皮细胞和 稳定过表达 CYP2J2 的血管平滑肌细胞系 检查 CYP2J2 产品对 Ca++ 信号传导的影响； (二) 共表达人 CYP2J2 和绿色荧光蛋白 培养新生大鼠心肌细胞以检查其作用 酶及其产物对基础条件下心肌细胞功能的影响 条件和代谢应激期间； (c) 构建转基因 在心肌中过度表达 CYP2J2 的小鼠来检查 CYP2J2 衍生的类二十烷酸对体内心脏功能的影响。 在相关研究中，我们鉴定了五个新的小鼠Cyp2j基因， 至少其中两个（Cyp2j6 和 Cyp2j8）似乎被表达 通过 RT-PCR 分析小鼠心脏。我们克隆了 相应的cDNA，并且正在表达 Sf9 昆虫细胞中的重组蛋白用于检查其 AA 代谢活动。初步研究表明 CYP2J6 血红素硫醇蛋白不稳定。这排除了酶促 CYP2J6蛋白分布的研究和分析 基于肽的抗体。我们目前正在研究方法 稳定重组 CYP2J6 以便这些研究将 可能的。