Arachidonic Acid Metabolism By Murine Cyp2c Isoforms

我们最近克隆了几个小鼠CYP 2C亚家族的新成员，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（ECO 2）和/或羟基二十碳四烯酸（HETE）中具有活性，所述类花生酸在许多组织中具有有效的生物活性。鼠类亚家族非常复杂（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。RT-PCR和免疫印迹研究表明，CYP 2C mRNA和蛋白质在肝和肝外组织中都很丰富，并且组织分布具有P450亚型特异性。我们最近鉴定了CYP 2C 29作为肺中主要的CYP 2C亚型，并证明它在气道上皮细胞中高度表达。我们已经克隆了Cyp 2c 29基因，其特征在于其内含子-外显子组织，并通过常规基因打靶策略产生Cyp 2c 29裸小鼠。将在基线和选定刺激（例如过敏原暴露、LPS暴露）后研究这些小鼠，以确定该P450在肺功能中的作用。我们还开发了一组针对鼠CYP 2C亚型的免疫特异性肽抗体，以便进一步研究它们的组织分布和调节。

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.