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活化。 第4类细胞色素催化白三烯、白藜芦醇和饱和脂肪酸的氧化。 4F亚家族成员既激活又抑制类花生酸的生物学功能。 人类表达CYP 4F 2和CYP 4F 3。 这两个基因都含有14个外显子和13个内含子。骨髓细胞表达一种形式的CYP 4F 3基因（A），其包含外显子4，而成人和胎儿肝脏表达另一种形式的基因CYP 4F 3B，其包含外显子3。 CYP 4F 2的已知形式包含外显子3。 CYP 4F 3A利用LTB 4而不是花生四烯酸作为底物，导致20-OH LTB 4的形成和该分子的生物活性失活。 CYP 4F 3B对LTB 4的KM比CYP 4F 3A高20倍以上。 通过与CYP 4F 3 B共享93%同一性的CYP 4F 2类比，CYP 4F 3 A和CYP 4F 3 B可能具有不同的底物特异性，其中CYP 4F 3 A使LTB 4失活，而CYP 4F 3 B从花生四烯酸产生生物活性代谢物20-HETE作为其优选反应。 我们提出，CYP 4F 3和CYP 4F 2的生物学作用是由协调的组织特异性表达和剪接各自的基因和mRNA决定的。 本提案的目的是确定CYP 4F 3和CYP 4F 2如何调节不同组织中20-HETE的形成和LTB 4的失活。 在第一个特定目标中，将通过使用亚型特异性抗体和半定量PCR确定CYP 4F 3B和CYP 4F 2对肝脏和肾脏中20-HETE形成的贡献。 在第二个特定目标中，将使用位点特异性诱变和计算机建模的组合来确定CYP 4F 3A和CYP 4F 3B之间底物特异性和KM变化的结构基础。在具体目标3中，将确定CYP 4F 3和CYP 4F 2的细胞特异性表达的基础。 荧光素酶报告基因构建体、DNA酶I足迹法、EMSA和诱变将鉴定决定造血细胞和肝细胞中CYP 4F 3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.