Arachidonic Acid Metabolism By Murine Cyp2c Isoforms

我们最近克隆了几个小鼠CYP2C亚家族的新成员，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）到环氧二十碳三烯酸（EETs）和/或羟基二十碳四烯酸（HETEs）的代谢中具有活性，二十碳三烯酸在许多组织中具有强大的生物活性。小鼠亚家族非常复杂（由15个基因和4个假基因组成）。尽管这些酶在氨基酸水平上是69-92%相同的，但它们在催化周转方面有显著差异，每种酶都有独特的产物谱。RT-PCR和免疫印迹研究显示，CYP2C mrna和蛋白在肝脏和肝外组织中均丰富，且组织分布具有P450亚型特异性。我们最近发现CYP2C29是肺中主要的CYP2C亚型，并证明它在气道上皮细胞中高度表达。我们克隆了Cyp2c29基因，对其内含子-外显子组织进行了表征，并通过传统的基因靶向策略产生了Cyp2c29无基因小鼠。这些小鼠将在基线和选择刺激（如过敏原暴露，LPS暴露）后进行研究，以确定P450在肺功能中的作用。我们还开发了一组基于免疫特异性肽的小鼠CYP2C亚型抗体，以便进一步研究其组织分布和调节。

We have recently cloned several new 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. The murine subfamily is very complex (consists of 15 genes and 4 pseudogenes). 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. RT-PCR and immunoblotting studies reveal that CYP2C mRNAs and proteins are abundant in both hepatic and extrahepatic tissues, and that the tissue distribution is P450 isoform-specific. We have recently identified CYP2C29 as the major CYP2C isoform in lung and demonstrated that it is highly expressed in airway epithelial cells. We have cloned the Cyp2c29 gene, characterized its intron-exon organization, and generated Cyp2c29 null mice by conventional gene targeting strategies. These mice will be studied at baseline and after selected stimuli (e.g. allergen exposure, LPS exposure) to determine the role of this P450 in lung function. We have also developed a panel of immunospecific peptide-based antibodies to the murine CYP2C isoforms so that their tissue distribution and regulation can be further investigated.