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: 6654612
• 负责人: BING K LAM
• 金额: $ 3.04万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6654612
• 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.

白细胞（LT）C4合酶是与谷胱甘肽（GSH）共轭LTA4（GSH）形成LTC4的关键酶，该酶是Cysteinyl Leukotrienes的母体化合物LTC4。因此，LTC4合酶可以通过其代谢物LTD4和LTE4来调节该有效介质的生物合成，从而有助于支气管哮喘的病理生物学。该项目的主要目的是使用报告基因构建体定义LTC4合酶基因的基因组调节（特定目标1）。另一个目标（特定目的2）是检查有或没有激活的白血病细胞中基因的调节，以及在成熟过程中未经转换的体外源自嗜酸性粒细胞中的调节。 LTA4的底物结合位点，蛋白质的膜拓扑以及涉及靶向其他研究区域的膜的信号（特定目标3）。跨作用因子将通过比较已鉴定的顺式作用元件与计算机数据库中已知因素的比较，以及通过凝胶移位和超级速度测定法进行比较。我们已经确定了一个550 bp的基因组片段，该片段由5'侧翼区域的66 bp的外显子1和484 bp组成，其中包含推定的引发剂元素和增强子元素。与其他无TATA的基因一样，假定的引发剂元素的SP-1结合位点44 bp 5'。这些调节元素将通过对瞬态转染中报告基因构建体的诱变分析进一步定义。通过DNase I超敏反应和在参与转录物表达增加的细胞中LTC4合酶的体外足迹，将寻求其他调节区域。 LTA4结合位点将通过对突变的重组蛋白的动力学分析来定义野生型酶的位置定向诱变。抗肽抗体将用于检查酶的亲水环，N末端和n末端的位置，以及与核包膜的外膜和核周膜相关的酶末端。 LTC4合酶的膜靶向信号将通过转染编码LTC4合酶肽片段绿色荧光蛋白（GFP）融合蛋白的cDNAS来寻求，然后转染荧光显微镜检查表达荧光融合蛋白的定位。