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Analysis of the processing mechanism of endothelin-converting enzyme and the development of its inhibitors as novel therapeutics for vascular complications.

分析内皮素转换酶的加工机制及其抑制剂作为血管并发症新疗法的开发。

基本信息

批准号：
08671167
负责人：
TAKAYANAGI Ryoichi
金额：
$ 1.47万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671167/
关键词：
Endothelin Endothelin-converting enzyme Endothelial cell Big endothelin ECE ET GFP

项目摘要

1. Big ET-1 analogues with the inhibitory activity on endothelin-converting enzyme-1 (ECE-1). Two big endothelin-1(big ET-1) analogues with the inhibitory activity on ECE-1, [F^<21>]big ET-1(18-34) and [A^<31>]big ET-1(18-34), were found by various amino acid substitution of big ET-1. Kinetics analyses showed that [F^<21>]big ET-1(18-34) is a competitive inhibitor and [A^<31>]big ET-1 (18-34) is a non-competitive inhibitor, suggesting that ECE-1 recognizes the P1 position and the C-terminal portion in a different fashon.2. Clarification of intracellular site for big ET-1-conversion. It is important for the drug design to know where in the cell the big ET-1 is converted, however, the intracellular site for the conversion of big ET-1 has been controversial. In the present study, using a chimera of ECE-1 and green fluorescent protein (GFP), visible signal in living cells, it was revealed that endogenous big ET-1 is converted intracellularly, not on the cell surface.3. Analyses of the properties of ECE-1 by mutagenesis of ECE-1 cDNA.Mutagenesis of human cDNA revealed that glycosylation of the extracellular C-terminal domain of ECE-1 is essential to the enzymatic activity, and that the transmembrane domain plays an important role in the completion of normal glycosylation.

1.BIG ET-1类似物具有抑制内皮素转换酶-1的活性。通过对大ET-1进行不同的氨基酸取代，发现了两个大的ET-1类似物，即[F^&lt；21&gt；]Big ET-1(18-34)和[A^&lt；31&gt；]Big ET-1(18-34)。动力学分析表明，大ET-1(18-34)是竞争性抑制物，而大ET-1(18-34)是非竞争性抑制物，这表明ECA-1以不同的方式识别P1位置和C-末端部分。阐明大的ET-1转化的胞内部位。对于药物设计来说，知道大ET-1在细胞中的哪里被转化是很重要的，然而，大ET-1转化的细胞内位置一直存在争议。在本研究中，利用活细胞中可见信号ECE1和绿色荧光蛋白(GFP)的嵌合体，揭示了内源性BIG-1是在细胞内转换的，而不是在细胞表面。通过对ECEs-1cDNAs的诱变，对ECEs-1的性质进行了分析。人的cDNAs突变表明，ECEs-1胞外C末端结构域的糖基化是酶活性所必需的，而跨膜结构域在完成正常的糖基化过程中起着重要作用。