Arachidonic acid metabolism by murine CYP2C isoforms

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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.