BIOCHEMICAL AND GENOMIC REGULATION OF HUMAN LTC SYNTHASE

白三烯（LT） C4合成酶是参与LTA4与谷胱甘肽（GSH）结合形成LTC4的关键酶，LTC4是半胱氨酸白三烯的母体化合物。因此，LTC4合成酶可以调节这种有效介质的生物合成，并通过其代谢物LTD4和LTE4参与支气管哮喘的病理生物学。该项目的主要目标是利用报告基因构建体确定LTC4合成酶基因的基因组调控（Specific Aim 1）。另一个目的（Specific Aim 2）是研究成熟过程中该基因在白血病细胞活化和未活化以及未转化的体外嗜酸性粒细胞中的调控作用。LTA4的底物结合位点、蛋白质的膜拓扑结构和膜靶向所涉及的信号是研究的额外领域（Specific Aim 3）。通过将已确定的顺式作用元素与计算机数据库中的已知因素进行比较，并通过凝胶移位和超移位测定来确定反式作用因素。我们已经确定了一个550-bp的基因组片段，由66 bp的外显子1和484 bp的5'侧区组成，包含一个假定的启动元件和增强元件。与其他TATA-less基因一样，在假定的启动元件的44 bp 5'处有一个SP-1结合位点。这些调控元件将通过瞬时转染中报告基因结构的诱变分析进一步确定。将通过Dnase I超敏试验和LTC4合成酶在参与转录物表达增加的细胞中的体外足迹来寻找其他调控区域。LTA4结合位点将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。抗肽抗体将用于检查亲水性环和酶的N端和C端相对于核膜的外膜和核周膜的位置。将LTC4合成酶肽片段-绿色荧光蛋白（GFP）融合蛋白编码的cdna转染到COS细胞中，通过荧光显微镜检测表达的荧光融合蛋白的定位，寻找LTC4合成酶的膜靶向信号。

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.