Regulation of intracellular proteolysis by calpastatin

钙蛋白酶抑制素是一种内源性抑制蛋白，特异性作用于钙蛋白酶（Ca^2+依赖性半胱氨酸蛋白酶）。钙蛋白酶抑制素与钙蛋白酶一起构成了与Ca^2+诱导的蛋白水解相关的细胞内调节系统。本研究计划为期三年（1985-1987），旨在阐明钙蛋白酶抑制素抑制钙蛋白酶的机制，特别是细胞内钙蛋白酶抑制素抑制钙蛋白酶的机制。以下是获得的主要结果。(1)免疫组织化学方法证明钙蛋白酶抑制蛋白在哺乳动物的各种组织中分布广泛，但有些不均匀。(2)两种不同的分子种类的钙蛋白酶抑制蛋白，显示68-和107-kDa的SDS-聚丙烯酰胺凝胶电泳迁移率，分离和表征。(3)猪钙蛋白酶抑制蛋白的分子克隆已经阐明了713个氨基酸残基的一级结构，其中包含四个重复结构域的结构。(4)在大肠杆菌中的表达大肠杆菌的cDNA的四个结构域已经揭示，每个结构域，具有约140个氨基酸残基，具有抑制活性的钙蛋白酶。使用定点诱变技术，从钙蛋白酶抑制蛋白单位结构域产生几个不同的片段，并比较它们的抑制效力。结果表明，calpastatin抑制calpain活性的关键部位是由约50个氨基酸残基组成的单位结构域的中心部分，本研究为calpastatin在体外对calpain的作用机制提供了基础信息，也为进一步阐明calpastatin在体内的作用机制提供了线索。

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.