BIOCHEMICAL AND GENOMIC REGULATION OF HUMAN LTC SYNTHASE

白三烯（LT）C4合酶是参与LTA 4与谷胱甘肽（GSH）结合形成半胱氨酰白三烯（Cys）母体化合物LTC 4的关键酶。因此，LTC 4合酶可以调节这种有效介质的生物合成，其通过其代谢产物LTD 4和LTE 4参与支气管哮喘的病理生物学。该项目的主要目的是使用报告构建体（特异性目标1）确定LTC 4合酶基因的基因组调控。另一个目的（具体目标2）是检查基因在白血病细胞中的调节，有和没有激活，并在非转化的体外衍生的嗜酸性粒细胞在成熟过程中。LTA 4的底物结合位点、蛋白质的膜拓扑结构和涉及膜靶向的信号是研究的额外领域（具体目标3）。反式作用因子将通过将鉴定的顺式作用元件与计算机数据库中的已知因子进行比较以及通过凝胶位移和超位移测定来鉴定。我们已经鉴定了一个550 bp的基因组片段，由66 bp的外显子1和484 bp的5'侧翼区组成，包含一个假定的起始元件和增强子元件。与其他TATA缺失基因一样，在推定的起始元件的5'端44 bp处存在SP-1结合位点。这些调控元件将通过瞬时转染中的报告构建体的诱变分析来进一步确定。将通过DNA酶I超敏性测定和参与增加的转录物表达的细胞中的LTC 4合酶的体外足迹法来寻找另外的调控区。通过野生型酶的定点诱变和突变重组蛋白的动力学分析来确定LTA 4结合位点。抗肽抗体将用于检查亲水环和N末端和C末端的酶相对于核膜的外膜和核周膜的位置。通过将编码LTC 4合酶肽片段-绿色荧光蛋白（GFP）融合蛋白的cDNA转染到COS细胞中，随后通过荧光显微镜检查表达的荧光融合蛋白的定位来寻找LTC 4合酶的膜靶向信号。

Leukotriene (LT) C4 synthase is the pivotal enzyme involved in conjugating LTA4 with glutathione (GSH) to form LTC4, the parent compound of the cysteinyl leukotrienes. Hence, LTC4 synthase can regulate the biosynthesis of this potent mediator, which contributes to the pathobiology of bronchial asthma through its metabolites, LTD4 and LTE4. The key objective of this project is to define genomic regulation of the LTC4 synthase gene using reporter constructs (Specific Aim 1). Another objective (Specific Aim 2) is to examine regulation of the gene in leukemic cells with and without activation and in non-transformed in vitro-derived eosinophils during maturation. The substrate binding site for LTA4, the membrane topology of the protein and the signal involved in membrane targeting re additional areas for study (Specific Aim 3). Trans-acting factors will be identify through comparison of the identified cis-acting elements with the known factors in a computer data base, and by gel shift and supershift assays. We have identified a 550-bp genomic fragment, composed of 66 bp of the exon 1 and 484 bp of 5' flanking region, that contains a putative initiator element and enhancer elements. Like other TATA-less genes, there is a SP-1 binding site 44 bp 5' of the putative initiator element. These regulatory elements will be further defined by mutagenic analysis of the reporter constructs in transient transfections. Additional regulatory regions will be sought by Dnase I hypersensitivity assays and in vitro footprinting of LTC4 synthase in cells engaged in increased transcript expression. The LTA4 binding site will be defined by site-directed mutagenesis of the wild-type enzyme with kinetic analysis of the mutated recombinant proteins. Anti-peptide antibodies will be used to examine the location of the hydrophilic loops and the N terminus and the C terminus of the enzyme in relation to the outer membrane and perinuclear membrane of the nuclear envelope. The membrane targeting signal of LTC4 synthase will be sought by transfection of cDNAs encoding for LTC4 synthase peptide fragment-green fluorescence protein (GFP) fusion proteins into COS cells followed by fluorescence microscopy examination of the localization of the expressed fluorescence fusion protein.