MOLECULAR ANALYSIS OF CYSTATHIONE BETA SYNTHASE DISORDERS IN HUMAN DISEASE

人类疾病中胱硫醚β合成酶紊乱的分子分析

• 批准号: 6108258
• 负责人: JAN P. KRAUS
• 金额: $ 19.43万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6108258
• 关键词: DNA footprinting; Escherichia coli; S adenosylmethionine; active sites; chemical binding; conformation; cystathionine beta synthase; enzyme activity; enzyme structure; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genotype; homocystinuria; human genetic material tag; human tissue; molecular pathology; phenotype; polymerase chain reaction; protein isoforms; protein structure function; site directed mutagenesis

The long-term objectives of this proposal are to define how abnormal expression of the cystathionine beta-synthase (CBS) gene plays a role in human disease, in particular, homocystinuria and vascular occlusive disease due to hyperhomocysteinemia. CBS, a central enzyme at a branch point in sulfur metabolism, is a tetramer of 63 kDa subunits; the enzyme binds two substrates, homocysteine and serine, and three ligands, PLP, AdoMet, and heme. It is encoded in humans on chromosome 21; its inherited deficiency causes the most common form of homocystinuria. Further, about one third of unselected patients with peripheral arterial disease reportedly are heterozygous for CBS deficiency. We isolated rat, mouse, and human CBS cDNA clones and their corresponding genes. We also showed that CBS pre- mRNA undergoes alternative splicing, yielding different enzyme isoforms. We cloned normal and mutant CBS cDNA into fusion expression vectors and developed methods to purify large amounts of the enzyme to homogeneity. We demonstrated that CBS is a heme-containing enzyme, and that the porphyrin is required for both PLP binding and activity; at least one mutations disrupts heme binding, resulting in B6-nonresponsive homocystinuria. We developed a rapid screening system and have identified 26 mutations in homocystinuria so far. Our specific aims are; 1) to provide detailed analysis of the human CBS gene; 2) to isolate and characterize the CBS promoter region; 3) to employ E. coli to prepare large amounts of CBS/beta-galactosidase fusion protein which can be subsequently cleaved yielding sufficient CBS for biochemical, biophysical and X-ray studies; 4) to determine the coenzyme pyridoxal; 5'phosphate and S-adenosylmethionine regions; and to define the catalytic center of the enzyme; 5) to investigate the amino acid sequence requirements for PLP, AdoMet, and heme binding through site-directed mutagenesis; and 6) to continue to locate and characterize mutations and to develop direct tests for frequent mutations. The role of this project is to extend our knowledge of the primary to the tertiary enzyme structure. This information is crucial to delineate disease manifestation and develop efficacious treatment paradigms, both by enzyme and, ultimately, gene therapy.

该提案的长期目标是定义异常情况如何 胱硫醚β-合酶（CBS）基因的表达在 人类疾病，特别是高胱氨酸尿症和血管闭塞性疾病 由于高同型半胱氨酸血症。 CBS，分支点的中心酶 硫代谢，是 63 kDa 亚基的四聚体；该酶结合两个 底物，同型半胱氨酸和丝氨酸，以及三种配体，PLP、AdoMet 和 血红素。 它被编码在人类的 21 号染色体上；它的遗传缺陷 引起最常见的高胱氨酸尿症。 此外，大约三分之一的 据报道，未选择的患有外周动脉疾病的患者 CBS 缺陷杂合子。 我们分离了大鼠、小鼠和人类 CBS cDNA 克隆及其相应基因。 我们还表明，哥伦比亚广播公司 (CBS) 预 mRNA 经历选择性剪接，产生不同的酶亚型。 我们将正常和突变的 CBS cDNA 克隆到融合表达载体中，并 开发了将大量酶纯化至均质的方法。 我们 证明 CBS 是一种含血红素的酶，并且卟啉 PLP 结合和活性都需要；至少一种突变 破坏血红素结合，导致 B6 无反应性同型半胱氨酸尿症。 我们 开发了快速筛查系统并鉴定出 26 个突变 到目前为止，同型半胱氨酸尿症。 我们的具体目标是； 1）提供详细的 人类CBS基因分析； 2) 分离和表征 CBS 启动子区； 3) 利用大肠杆菌制备大量 CBS/β-半乳糖苷酶融合蛋白，随后可被切割 产生足够的 CBS 用于生化、生物物理和 X 射线研究； 4） 测定辅酶吡哆醛； 5'磷酸和S-腺苷甲硫氨酸 地区；并确定酶的催化中心； 5）到 研究 PLP、AdoMet 和血红素的氨基酸序列要求 通过定点诱变结合； 6) 继续寻找和 表征突变并开发针对频繁突变的直接测试。 这个项目的作用是将我们对初级的知识扩展到 三级酶结构。 此信息对于描绘至关重要 疾病表现并开发有效的治疗范例 酶，最终是基因疗法。