Arachidonic acid metabolism by murine CYP2C isoforms

我们克隆了小鼠CYP 2C亚家族的5个成员，并在大肠杆菌中表达了重组P450蛋白。大肠杆菌中，并显示它们在花生四烯酸（AA）代谢为环氧二十碳三烯酸（EEE）和/或羟基二十碳四烯酸（HETE）中具有活性，类二十烷酸在包括肺和肠的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，但它们的催化周转率明显不同，并且每种酶都有独特的产物谱。例如，CYP 2C 29生物合成14，15-EET，而CYP 2C 37（与CYP 2C 29有77%的相同性）主要合成12-HETE。CYP 2C 40是不寻常的，因为它产生16-HETE。此外，鼠CYP 2C酶以高度区域选择性和立体选择性的方式产生雌二醇。这些不同的催化剂之间的高度同源性 酶为研究结构-活性关系提供了独特的机会。我们将根据CYP 2C 5（一种已结晶的哺乳动物P450）的晶体结构，对鼠CYP 2C酶的活性位点进行建模。建模将有助于预测哪些氨基酸可能是负责的催化多样性的小鼠CYP 2C，并将用于计划嵌合和诱变实验。RT-PCR和免疫印迹数据表明，小鼠CYP 2C mRNA和蛋白质在肝和肝外组织中都是丰富的，并且组织分布是P450亚型特异性的。有趣的是，CYP 2C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产生的PCR产物进行测序，鉴定了肺（CYP 2C 29）和结肠（CYP 2C 40）中的主要CYP 2C亚型。CYP 2C 29和CYP 2C 40分别在肺和肠内的细胞定位将提供关于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CYP 2C过表达对肺和肠细胞功能的影响。将CYP 2C 29和CYP 2C 40 cDNA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、细胞增殖和细胞凋亡等参数的影响。最后，我们将产生缺乏Cyp 2c 29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在肺中的已知生物学效应。

We have recently cloned five members of the mouse 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 including the lung and intestine. 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, whereas CYP2C37 (which is 77% identical to CYP2C29) primarily makes 12-HETE. CYP2C40 is unusual in that it produces 16-HETE. Moreover, the murine CYP2C enzymes produce EETs in a highly regio- and stereoselective fashion. The high degree of homology between these catalytically distinct enzymes affords a unique opportunity to study structure-activity relationships. We will model the active sites of the murine CYP2C enzymes based on the crystal structure of CYP2C5, a mammalian P450 that has been crystalized. The modeling will help to predict which amino acids are likely to be responsible for the catalytic diversity of the murine CYP2Cs and will be used to plan chimeragenesis and mutagenesis experiments. 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, will 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.