Studies on mechanisms of calpain activation in the cell

钙蛋白酶是一种“调节蛋白酶”，参与细胞功能所必需的Ca^2+介导的信号转导过程。钙蛋白酶的生理功能虽然还不清楚，但它是如此重要，以致钙蛋白酶活性不足会导致各种疾病，包括肌营养不良症、缺血性疾病、癌症、糖尿病和阿尔茨海默病。在这些疾病中，抑制钙蛋白酶活性经常被报道可以改善患者的病理状态，从而表明钙蛋白酶抑制剂和理解钙蛋白酶激活机制的重要性。在这项研究中，我们表达了相应的蛋白酶结构域的钙蛋白酶和EGFP标记的C105 S显性负m-钙蛋白酶的重组蛋白，并用于研究钙蛋白酶活性的调节的分子机制。令我们惊讶的是，m-钙蛋白酶的蛋白酶结构域显示出Ca^2+依赖性的蛋白酶活性，这表明蛋白酶结构域本身与Ca^2+结合。这是关于钙蛋白酶活性如何调节的一个非常重要的信息。此外，使用C105 S-显性至阴性m-钙蛋白酶，细胞骨架蛋白如fodrin和talin的蛋白水解显示出对m-和μ-钙蛋白酶的不同敏感性。钙蛋白酶抑制素是钙蛋白酶的高度特异性和强有力的内源性抑制蛋白，它不抑制这些蛋白的蛋白水解。这些结果有力地表明，对于钙蛋白酶的蛋白水解及其调节，膜，而不是钙蛋白酶抑制剂，是非常重要的因素。

Calpain is a "modulator protease" involved in Ca^<2+>-mediated signal transduction processes essential for cellular functions. Physiological functions, although yet unclear, of calpains are so important that a deficient calpain activity causes various diseases including muscular dystrophies, ischemia diseases, cancers, diabetes, and Alzheimer's disease. In these diseases, inhibition of calpain activity is often reported to improve the pathological states of patients, thus indicating the importance of calpain inhibitors and understanding of mechanisms of calpain activation. In this study, we expressed recombinant proteins corresponding to the protease domain of calpain and to EGFP-tagged C105S dominant negative m-calpain, and used to study molecular mechanisms of the regulation of calpain activity. To our surprise, the protease domain of m-calpain showed Ca^<2+>-dependent protease activity, indicating that the protease domain itself binds Ca^<2+>. This is a very important information as to how calpain activity is regulated. Moreover, using C105S-dominanto negative m-calpain, proteolysis of cyteskeletal proteins such as fodrin and talin showed differential susceptibility to m- and μ-calpains. Calpastatin, which is the highly specific and powerful endogenous inhibitor protein for calpain, did not suppress proteolysis of these proteins. These results strongly suggest that for proteolysis of calpain and its regulation, membrane, rather than calpastatin, is very important factor.