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

细胞中的每一种蛋白质都有自己的半衰期，从几分钟到几周不等。蛋白质的寿命长短是由以calpain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，在细胞中调节信号转导系统。它对参与信号转导系统的其他蛋白质的特定位点进行蛋白水解，以改变其活性状态和/或结构。在骨骼肌中，除了普遍表达的常规钙蛋白酶、μ-钙蛋白酶和m-钙蛋白酶外，还存在一种叫做p94的肌肉特异性钙蛋白酶。P94具有快速、彻底的自溶活性，体外半衰期极短。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在体内通过与连接体的结合而稳定。2) p94具有4个结构域结构，特异的插入区IS1和IS2参与了p94的快速自溶。如果IS1或IS2被删除，p94的表达稳定。3)在骨骼肌中，p94的备选剪接产物以特定阶段的方式表达。部分具有IS1和/或IS2缺失，具有稳定的蛋白水解活性。4)令我们惊讶的是，p94优先切割calpastatin，这是传统calpain的内源性特异性抑制蛋白。这些结果提示p94与常规钙蛋白酶协同作用，是骨骼肌细胞蛋白转换系统的关键调节因子。

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.