STRUCTURAL DETERMINANTS OF BIG ENDOTHELIN-1 PROCESSING

大内皮素-1 加工的结构决定因素

• 批准号: 2835624
• 负责人: ADVIYE ERGUL
• 金额: $ 4.43万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-22 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2835624
• 关键词: CHO cells; endopeptidases; endothelin; enzyme activity; enzyme structure; hormone receptor; neuropeptide receptor; nucleic acid sequence; protease inhibitor; protein structure function; receptor sensitivity; site directed mutagenesis; transfection; zymogens

Epidemiologic studies indicate that death from cardiovascular disease is the number one cause of natural deaths in this country. It is well established that the endothelium is very important in the control of cardiovascular homeostasis in health and disease. Factors released from endothelial cells modulate the vascular smooth muscle contractility and proliferation, which are altered in some disease states such as hypertension and atherosclerosis. One of these factors, endothelin-1 (ET-1), has been shown to be involved in systemic and pulmonary hypertension, advanced atherosclerosis, and acute myocardial infarction. The elevated plasma ET-1 levels in these patients can be regulated at the biosynthesis level by blocking the conversion of big ET-1 to bioactive ET-1 with endothelin converting enzyme (ECE) inhibitors and/or at the receptor level using receptor antagonists. Although much work has been done on endothelin receptor antagonists and on the purification and cloning of ECE, the structural determinants required for the high specificity of ECE-1 for big ET-1 is not known. One approach is to study the contribution of conserved amino acid residues or specific domains to the structure of big ET-1 and ECE-1. In this research proposal, highly conserved amino acid residues in big ET-1 will be replaced by the corresponding residues in big ET-2 and big ET-3 using site-directed mutagenesis of preproendothelin-1 (PPET-1) cDNA. The mutant or wild-type cDNAs and ECE-1 cDNA will then be co-transfected into Chinese hamster ovary (CHO) cells. The effects of these mutations on the conversion of big ET-1 to ET-1 by ECE-1 will be tested by analyzing the transfection media for immunoreactive big ET-1 and ET-1, and the results will be compared to that of wild-type big ET- 1. In addition, a truncated ECE- 1 cDNA will be constructed by replacing the sequence of the ECE- 1 cDNA encoding the N-terminal region of ECE- 1, including the membrane-spanning region (amino acid residues 1-77 in ECE-1), by the secretion signal sequence of PPET-1 and an affinity tag of six consecutive histidine residues using polymerase chain reaction. The wild-type and truncated ECE-1 proteins will be expressed in CHO cells. The His-tagged soluble enzyme will be purified using nickel-nitrilotriacetate (Ni-NAT) columns and soluble membrane fractions containing the native enzyme will be prepared. The recombinant native membrane-bound and soluble ECE-1 will then be characterized by analyzing the conversion of synthetic big ET-1 to ET-1 in vitro. The proposed studies using current techniques in molecular biology will provide information on the structure of ECE-1, as well as developing new approaches to prepare increased amounts of enzyme for structural studies and antibody production. The results of this study have the potential to lead to the development of new ECE inhibitors which could be used as therapeutic agents.

流行病学研究表明，死于心血管疾病的人数 是这个国家自然死亡的头号原因公 确定了内皮细胞在控制 健康和疾病中的心血管稳态。释放的因素 内皮细胞调节血管平滑肌收缩性， 在某些疾病状态下会发生改变， 高血压和动脉粥样硬化。 这些因素之一，内皮素-1 ET-1参与了全身和肺部的炎症反应。 高血压、晚期动脉粥样硬化和急性心肌梗死。 这些患者血浆ET-1水平的升高可以通过调节血浆ET-1水平来调节。 通过阻断大ET-1向生物活性物质转化， ET-1与内皮素转换酶（ECE）抑制剂和/或在 受体水平使用受体拮抗剂。 尽管已经对内皮素受体拮抗剂和内皮素受体拮抗剂进行了大量的研究， 关于ECE的纯化和克隆，结构决定因素 ECE-1对大ET-1的高特异性所需的条件尚不清楚。一 方法是研究保守氨基酸残基的贡献 或大ET-1和ECE-1结构的特定结构域。在这 研究建议，大ET-1中高度保守的氨基酸残基将 用大ET-2和大ET-3中的相应残基替换， 内皮素原-1（PPET-1）cDNA定点突变。突变 或将野生型cDNA和ECE-1 cDNA共转染入 中国仓鼠卵巢（CHO）细胞。这些突变对 通过分析ECE-1将大ET-1转换为ET-1， 免疫反应性大ET-1和ET-1的转染培养基，结果 将其与野生型大ET- 1进行比较。此外，一个截断的 通过替换ECE- 1的序列，将构建ECE- 1 cDNA。 编码ECE- 1的N-末端区域的cDNA，包括 跨膜区（ECE-1中的氨基酸残基1-77），通过 PPET-I的分泌信号序列和六个的亲和标签 连续的组氨酸残基，使用聚合酶链反应。的 野生型和截短的ECE-1蛋白将在CHO细胞中表达。 His标记的可溶性酶将使用 次氮基三乙酸镍（Ni-NAT）柱和可溶性膜级分 将制备含有天然酶的混合物。重组天然 膜结合的和可溶的ECE-1然后将通过分析 体外合成的大ET-1转化为ET-1。拟议 利用当前分子生物学技术的研究将提供 欧洲经委会第一次会议的结构资料， 制备用于结构研究的增加量的酶的方法 和抗体生产。这项研究的结果有可能 导致开发新的ECE抑制剂，可用作 治疗剂。