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Research on molecular mechanisms of muscle protein turn-over by structure-function relationship studies of calpain

通过钙蛋白酶的结构-功能关系研究肌肉蛋白质周转的分子机制

基本信息

批准号：
13660119
负责人：
SORIMACHI Hiroyuki
金额：
$ 2.37万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Every protein in the cell turns over with its own half-life, spanning from a few minutes to several weeks. The length of the lifetime of protein is determined by intracellular proteolytic system represented by "calpain". Calpain is a calcium-requiring cysteine protease, which modulate signal transduction system in the cell. It proteolyzes a specific site of other proteins involved in signal transduction system to change their state of activity and/or structure. In skeletal muscle, a muscle-specific calpain called p94 functions in addition to the ubiquitously expressed conventional calpains, μ- and m-calpains. p94 has very short half-life in vitro due to its rapid and exhaustive autolytic activity. In this study, we investigate a behavior of p94 in skeletal muscle to elucidate cellular proteolytic system that modulates various proteins' lifetime. As a result, the followings have been revealed : 1) p94 is stabilized by binding to connetin in vivo. 2) p94 has 4 domain structure, and the specific insertion regions, IS1 and IS2, are involved in the rapid autolysis of p94. If either IS1 or IS2 is deleted, p94 shows stable expression. 3) In skeletal muscle, alternative splicing products of p94 are expressed in a stage-specific manner. Some of them have deletion in IS1 and/or IS2, showing stable proteolytic activity. 4) To our surprise, p94 preferentially cuts calpastatin, which is the endogenous specific inhibitor protein for the conventional calpains. These results suggested p94 in cooperation with the conventional calpains functions key modulator of protein turnover system in skeletal muscle cells.

细胞中的每种蛋白质都有自己的半衰期，从几分钟到几周不等。蛋白质寿命的长短是由以“钙蛋白酶”为代表的细胞内蛋白水解系统决定的。钙蛋白酶是一种需要钙的半胱氨酸蛋白酶，调节细胞内的信号转导系统。它可以水解参与信号转导系统的其他蛋白质的特定位点，以改变其活性和/或结构状态。在骨骼肌中，一种称为p94的肌肉特异性钙蛋白酶除了普遍表达的常规钙蛋白酶μ-和m-钙蛋白酶外还发挥作用。由于其快速和彻底的自溶活性，p94在体外具有非常短的半衰期。在这项研究中，我们研究了p94在骨骼肌中的行为，以阐明调节各种蛋白质寿命的细胞蛋白水解系统。结果表明：1）p94在体内通过与连接蛋白结合而稳定。2)p94具有4个结构域，其特异性插入区域IS 1和IS 2参与p94的快速自溶。如果缺失IS 1或IS 2，则p94显示稳定表达。3)在骨骼肌中，p94的选择性剪接产物以阶段特异性方式表达。其中一些在IS 1和/或IS 2中存在缺失，表现出稳定的蛋白水解活性。4)令人惊讶的是，p94优先切割钙蛋白酶抑制蛋白，这是一种内源性的特异性抑制蛋白的传统钙蛋白酶。这些结果提示p94与钙蛋白酶协同作用是骨骼肌细胞蛋白质周转系统的关键调节因子。