PHARMACOLOGY OF 4F CYTOCHROME P450'S

细胞色素P450催化药物和天然产物的代谢，导致其生物学特性的失活或激活。第四类细胞色素催化白三烯、前列腺素和饱和脂肪酸的氧化。4F亚家族成员具有激活和失活二十烷类化合物的生物学功能。人类表达细胞色素P4F2和细胞色素P4F3。这两个基因都含有14个外显子和13个内含子。髓系细胞表达一种形式的含有外显子4的CYP4F3基因(A)，而成人和胎儿肝脏表达另一种形式的基因，即含有外显子3的CYP4F3B。已知的形式的CYP4F2含有外显子3。CYP4F3A利用LTB4，但不利用花生四烯酸作为底物，导致20-OH LTB4的形成和该分子的生物活性失活。CYP4F3B，LTB4的KM比CYP4F3A高20倍以上。与与CYP4F3B有93%同源性的CYP4F2类似，CYP4F3A和CYP4F3B可能具有不同的底物专一性，其中CYP4F3A使LTB4失活，而CYP4F3B从花生四烯酸生成具有生物活性的代谢物20-HETE是其首选反应。我们认为，CYP4F3和CYP4F2的生物学作用是由各自基因和mRNAs在组织中的协调表达和剪接决定的。这项建议的目的是确定CYP4F3和CYP4F2如何调节不同组织中20-HETE的形成和LTB4的失活。在第一个特定目标中，将通过使用异构体特异性抗体和半定量PCR来确定CYP4F3B和CYP4F2对肝和肾中20-HETE形成的贡献。在第二个特定目标中，将结合定点突变和计算机模拟来确定CYP4F3A和CYP4F3B之间底物专一性和Km变化的结构基础。在具体目标3中，将确定细胞特异性表达CYP4F3和CYP4F2的基础。荧光素酶报告构建、DNase I足迹、EMSA和突变将确定决定CYP4F3在造血细胞和肝细胞中表达的启动子元件。

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.