Arachidonic Acid Metabolism By Murine CYP2C Isoforms

小鼠 CYP2C 亚型的花生四烯酸代谢

• 批准号: 6546715
• 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/6546715
• 关键词: Escherichia coli; apoptosis; arachidonate; cell line; cell proliferation; cytochrome P450; eicosanoid metabolism; enzyme activity; enzyme biosynthesis; enzyme mechanism; gene induction /repression; genetically modified animals; ion transport; laboratory mouse; large intestine; lung; messenger RNA; molecular cloning; polymerase chain reaction; protein isoforms; protein localization; tissue /cell culture; western blottings

We have recently cloned five members of the murine CYP2C subfamily, expressed the recombinant P450 proteins in E. coli, and showed that they are active in the metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) and/or hydroxyeicosatetraenoic acids (HETEs), eicosanoids that possess potent biological activities in numerous tissues. Despite the fact that these enzymes are 69-92% identical at the amino acid level, they differ markedly in their catalytic turnover and each enzyme has a unique product profile. For example, CYP2C29 biosynthesizes 14,15-EET, CYP2C37 primarily makes 12-HETE, and CYP2C40 produces 16-HETE. Moreover, the murine CYP2C enzymes produce EETs in a highly regio- and stereoselective fashion. RT-PCR and immunoblot data indicate that the mouse CYP2C mRNAs and proteins are abundant in both hepatic and extrahepatic tissues, and that the tissue distribution is P450 isoform-specific. Interestingly, the CYP2C proteins are present in high concentrations in the lung and intestine. We have recently identified the major CYP2C isoform in lung (CYP2C29) and colon (CYP2C40) by sequencing PCR products generated using universal primers. The cellular localization of CYP2C29 and CYP2C40 within lung and intestine, respectively, provide further clues regarding the functional role of these enzymes in these tissues. We will develop in vitro systems to study the effects of CYP2C overexpression on lung and intestinal cell function. The CYP2C29 and CYP2C40 cDNAs will be transfected into cultured mouse lung and intestinal cell lines, respectively, to examine effects on parameters such as ion transport, cellular proliferation and apoptosis. Finally, we will generate mice that lack the Cyp2c29 gene. This P450 was chosen because of its tissue distribution and the known biological effects of its major product, 14,15-EET, in the lung.

我们最近克隆了小鼠 CYP2C 亚家族的五个成员，在大肠杆菌中表达了重组 P450 蛋白，并表明它们在花生四烯酸（AA）代谢为环氧二十碳三烯酸（EET）和/或羟基二十碳四烯酸（HETE）中具有活性），类二十烷酸在许多组织中具有有效的生物活性。尽管这些酶在氨基酸水平上有 69-92% 的相同性，但它们的催化转化率显着不同，并且每种酶都有独特的产物特征。例如，CYP2C29 生物合成 14,15-EET，CYP2C37 主要生成 12-HETE，CYP2C40 生成 16-HETE。此外，小鼠 CYP2C 酶以高度区域和立体选择性的方式产生 EET。 RT-PCR 和免疫印迹数据表明，小鼠 CYP2C mRNA 和蛋白质在肝组织和肝外组织中丰富，并且组织分布具有 P450 亚型特异性。有趣的是，CYP2C 蛋白在肺和肠中以高浓度存在。我们最近通过对使用通用引物生成的 PCR 产物进行测序，鉴定了肺 (CYP2C29) 和结肠 (CYP2C40) 中的主要 CYP2C 亚型。 CYP2C29 和 CYP2C40 分别在肺和肠内的细胞定位提供了有关这些酶在这些组织中的功能作用的进一步线索。我们将开发体外系统来研究 CYP2C 过度表达对肺和肠细胞功能的影响。 CYP2C29 和 CYP2C40 cDNA 将分别转染到培养的小鼠肺和肠细胞系中，以检查对离子转运、细胞增殖和凋亡等参数的影响。最后，我们将产生缺乏 Cyp2c29 基因的小鼠。选择 P450 是因为其组织分布及其主要产物 14,15-EET 在肺部的已知生物效应。