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Regulation of intracellular proteolysis by calpastatin

钙蛋白酶抑制剂对细胞内蛋白水解的调节

基本信息

批准号：
60440031
负责人：
MURACHI Takashi
金额：
$ 14.53万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (Ca^<2+>-dependent cysteine proteinases). With calpain, calpastatin constitutes an intracellular regulatory system as related to Ca^<2+>-induced proteolysis. The present three-year project for 1985-1987 aimed at the elucidation of the mechanism of inhibition of calpain by calpastatin, particularly that occurring inside a cell. The followings are the major results obtained.(1) Very wide, but somewhat uneven, distribution of calpastatin in various mammalian tissues has been demonstrated by immunohistochemical methods.(2) Two different molecular species of calpastatin, showing 68- and 107-kDa mobilities on SDS-Polyacrylamide gel electrophoresis, were isolated and characterized.(3) Molecular cloning of pig calpastatin has led to the elucidation of the primary structure of 713-amino acid residues, which contained a four-repetitive-domain structure.(4) Expression in E. coli of cDNAs for the four domains has revealed that each domain, having approximately 140 amino acid residues, possesses inhibitory activity against calpain. Using techniques for site-directed mutagenesis, several different fragments were created from a calpastatin unit domain, and they were compared with respect to inhibitory potency. It was thus concluded that a central portion of a unit domain, composed of some 50 amino acid residues, is essential for the inhibition of calpain activity.The present study has provided fundamental information as to the in vitro mechanism of calpastatin action on calpain, and it has also given some clue how to elucidate the mechanism of its action in vivo.

钙调蛋白是一种内源性抑制蛋白，特异性作用于钙蛋白酶(钙依赖的半胱氨酸蛋白酶)。在钙蛋白酶的作用下，钙调蛋白构成了一个与钙离子诱导的蛋白分解有关的细胞内调控系统。本项为期三年的1985-1987年计划旨在阐明钙调蛋白抑制钙蛋白酶的机制，特别是发生在细胞内的机制。获得的主要结果如下：(1)免疫组织化学方法显示Calastatin在哺乳动物组织中的分布非常广泛，但有一定的不均一性。(2)分离并鉴定了两种不同的分子物种，在SDS-PAGE上显示出68 kDa和107 kDa的迁移率。(3)猪Calastatin的分子克隆揭示了713个氨基酸残基的一级结构，它包含一个四个重复的结构域结构。(4)在大肠杆菌中表达的这四个结构域的cDNAs表明，每个结构域都有大约140个氨基酸残基，具有抑制钙痛的活性。利用定点突变技术，从钙调蛋白单位结构域中创建了几个不同的片段，并比较了它们的抑制效力。本研究为钙蛋白酶的体外作用机制提供了基础信息，也为阐明其在体内的作用机制提供了一些线索。