PHARMACOLOGY OF 4F CYTOCHROME P450'S

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Cytochrome P450s catalyze the metabolism of drugs and natural products, resulting in either the inactivation or activation of their biological properties. The class 4 cytochromes catalyze the oxidation of leukotrienes, prostaglandins, and saturated fatty acids. The 4F subfamily members both activate and inactivate eicosanoids for biological function. Humans express CYP4F2 and CYP4F3. Both genes contains 14 exons and 13 introns. Myeloid cells express one form of the CYP4F3 gene(A) that incorporates exon 4, whereas adult and fetal liver express another form of the gene, CYP4F3B, that incorporates exon 3. The known form of CYP4F2 incorporates exon 3. CYP4F3A utilizes LTB4, but not arachidonic acid as a substrate, resulting in the formation of 20-OH LTB4 and the inactivation of this molecule for biological activity. CYP4F3B, has a KM for LTB4 that is over 20- times higher than that of CYP4F3A. By analogy with CYP4F2, which shares 93 percent identity with CYP4F3B, CYP4F3A and CYP4F3B likely possess distinct substrate specificity, with CYP4F3A inactivating LTB4, and CYP4F3B generating the bioactive metabolite 20-HETE from arachidonic acid as its preferred reaction. We propose that the biological roles of CYP4F3 and CYP4F2 are determined by the coordinated tissue-specific expression and splicing of the respective genes, and mRNAs. The goal of this proposal is to determine how CYP4F3 and CYP4F2 regulate the formation of 20-HETE and the inactivation of LTB4 in different tissues. In the first specific aim the contribution of CYP4F3B and CYP4F2 to 20-HETE formation in liver and kidney will be determined by the use of isoform specific antibodies, and semiquantititative PCR. In the second specific aim, the structural basis for the change in substrate specificity and KM between CYP4F3A and CYP4F3B will be determined using a combination of site specific mutagenesis and computer modeling. In specific aim 3, the of basis of cell-specific expression of CYP4F3 and CYP4F2 will be determined. Luciferase reporter constructs, DNase I footprinting, EMSA, and mutagenesis will identify promoter elements determining the expression of CYP4F3 in hematopoetic and liver cells.