STRUCTURAL DETERMINANTS OF BIG ENDOTHELIN 1 PROCESSING

大内皮素 1 加工的结构决定因素

• 批准号: 2764011
• 负责人: ADVIYE ERGUL
• 金额: $ 3.6万
• 依托单位: UNIVERSITY OF GEORGIA
• 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/2764011
• 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 这是这个国家自然死亡的头号原因。这很好 确定内皮细胞在控制血管紧张性心脏病中非常重要 健康和疾病中的心血管动态平衡。释放的因子 血管内皮细胞调节血管平滑肌的收缩能力 增殖，在某些疾病状态下会发生变化，如 高血压和动脉粥样硬化。其中一种因子是内皮素-1 (ET-1)，已被证明参与全身和肺 高血压、晚期动脉粥样硬化和急性心肌梗死。 这些患者升高的血浆ET-1水平可以在 阻止BIG ET-1向生物活性转化的生物合成水平 ET-1与内皮素转换酶(ECEs)抑制剂和/或 受体水平使用受体拮抗剂。 尽管在内皮素受体拮抗剂和药物方面已经做了很多工作 结构决定子ECEs的纯化和克隆 ECE1对大ET-1的高度特异性所需的作用尚不清楚。一 方法是研究保守氨基酸残基的贡献 或特定结构域与BIG ET-1和ECE1的结构有关。在这 研究建议，大ET-1中高度保守的氨基酸残基将 用BIG ET-2和BIG ET-3中的相应残基取代 PPET-1基因的定点突变。变种人 或野生型cDNAs与ECE1的cDNAc DNA共转染 中国仓鼠卵巢(CHO)细胞。这些突变对细胞的影响 欧洲经委会-1将测试BIG ET-1到ET-1的转换，方法是分析 免疫活性BIG ET-1和ET-1的转染液及其结果 将与野生型BIG-1进行比较。此外，一个截短的 将Ece-1的序列替换为Ece-1的序列，构建Ece-1的cDNA 编码ECE1 N-末端区域的基因，包括 膜跨区(ECE1中的氨基酸残基1-77)，由 PPET-1分泌信号序列及6个亲和标签 用聚合酶链式反应检测连续组氨酸残基。这个 野生型和截短型ECE1蛋白将在CHO细胞中表达。 His标记的可溶酶将使用 镍-三乙酸氮酯(Ni-NAT)色谱柱和可溶性膜组分 将制备含有天然酶的产品。重组原生生物 膜结合的和可溶的ECE1随后将通过分析 人工合成的BIG-1体外转化为ET-1。建议数 利用分子生物学现有技术的研究将提供 关于欧洲经委会-1的结构的信息，以及开发新的 用于结构研究的增加酶的制备方法 和抗体的产生。这项研究的结果有可能 导致开发新的欧洲经委会抑制剂，可用于 治疗剂。