BIOCHEMICAL AND GENOMIC REGULATION OF HUMAN LTC SYNTHASE

人类 LTC 合成酶的生化和基因组调控

• 批准号: 6202646
• 负责人: BING K LAM
• 金额: $ 32.71万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202646
• 关键词: DNA footprinting; binding sites; cell line; chimeric proteins; eicosanoid metabolism; enzyme structure; fluorescence microscopy; genetic mapping; genetic promoter element; genetic regulation; glutathione; glutathione transferase; green fluorescent proteins; human tissue; leukotrienes; site directed mutagenesis; transcription factor

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.

白三烯 (LT) C4 合酶是参与 LTA4 与谷胱甘肽 (GSH) 结合形成 LTC4（半胱氨酰白三烯的母体化合物）的关键酶。因此，LTC4 合酶可以调节这种有效介质的生物合成，通过其代谢物 LTD4 和 LTE4 促进支气管哮喘的病理生物学。该项目的主要目标是使用报告构建体来定义 LTC4 合酶基因的基因组调控（具体目标 1）。另一个目标（具体目标 2）是检查激活和未激活的白血病细胞以及成熟过程中未转化的体外来源的嗜酸性粒细胞中该基因的调节。 LTA4 的底物结合位点、蛋白质的膜拓扑结构以及参与膜靶向的信号是其他需要研究的领域（具体目标 3）。通过将已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，并通过凝胶位移和超位移测定来鉴定反式作用因子。我们鉴定了一个 550 bp 的基因组片段，由 66 bp 的外显子 1 和 484 bp 的 5' 侧翼区域组成，其中包含假定的起始元件和增强子元件。与其他无 TATA 的基因一样，在推定起始元件的 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构建体的诱变分析进一步确定。通过 Dnase I 超敏试验和参与增加转录物表达的细胞中 LTC4 合酶的体外足迹分析，将寻找其他调控区域。 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定义。抗肽抗体将用于检查亲水环以及酶的 N 末端和 C 末端相对于核膜的外膜和核周膜的位置。通过将编码LTC4合酶肽片段-绿色荧光蛋白(GFP)融合蛋白的cDNA转染至COS细胞中，然后通过荧光显微镜检查所表达的荧光融合蛋白的定位来寻找LTC4合酶的膜靶向信号。