STRUCTURAL DETERMINANTS OF BIG ENDOTHELIN-1 PROCESSING

流行病学研究表明，心血管疾病导致的死亡 该国自然死亡的第一大原因。很好 确定内皮细胞在控制中非常重要 健康和疾病中的心血管稳态。释放因素 内皮细胞调节血管平滑肌收缩力 增殖，在某些疾病状态下会发生改变，例如 高血压和动脉粥样硬化。 这些因素之一，内皮素-1 (ET-1)，已被证明参与全身和肺部 高血压、晚期动脉粥样硬化和急性心肌梗塞。 这些患者升高的血浆 ET-1 水平可以通过以下途径进行调节： 通过阻断大 ET-1 转化为生物活性物质来提高生物合成水平 ET-1 与内皮素转化酶 (ECE) 抑制剂和/或 使用受体拮抗剂的受体水平。 尽管内皮素受体拮抗剂和 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结构的特定域。在这个 研究提案，大ET-1中高度保守的氨基酸残基将 使用 big ET-2 和 big ET-3 中的相应残基替换 前内皮素原 1 (PPET-1) cDNA 的定点诱变。突变体 或野生型 cDNA 和 ECE-1 cDNA 将共转染到 中国仓鼠卵巢 (CHO) 细胞。这些突变的影响 ECE-1 将大 ET-1 转换为 ET-1 将通过分析 免疫反应性大 ET-1 和 ET-1 的转染培养基及其结果 将与野生型大 ET-1 进行比较。此外，截短的 ECE-1 cDNA将通过替换ECE-1的序列来构建 编码 ECE-1 N 末端区域的 cDNA，包括 跨膜区域（ECE-1 中的氨基酸残基 1-77），由 PPET-1 的分泌信号序列和 6 个亲和标签 使用聚合酶链式反应分析连续的组氨酸残基。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纯化 次氮基三乙酸镍 (Ni-NAT) 柱和可溶性膜组分 将制备含有天然酶的。重组天然 然后通过分析来表征膜结合和可溶性 ECE-1 合成大ET-1在体外转化为ET-1。拟议的 使用分子生物学现有技术的研究将提供 有关 ECE-1 结构的信息，以及开发新的 制备更多酶用于结构研究的方法 和抗体的产生。这项研究的结果有可能 导致新的 ECE 抑制剂的开发，该抑制剂可用作 治疗剂。

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.