Analysis of molecular mechanism of muscular dystrophy involving skeletal muscle-specific calpain and connectin

钙蛋白酶是一种典型的细胞内钙依赖性蛋白酶，能以有限的方式水解各种细胞蛋白，改变其性质，是一种生物调节剂。P94是骨骼肌中特异性表达的钙蛋白酶同源物。p94具有不同于普通钙蛋白的各种特性，例如，在缺乏Ca2+的情况下非常迅速地自溶，至少在两个特定的位点与骨骼肌连接蛋白结合，包含核易位信号，有三个插入序列，水解fodrin， calpastatin， hsp60等。最近，p94被确定为肢体带型2A肌营养不良症（LGMD2A）的基因。我们从LGMD2A患者中发现的各种突变体中选择了10个点突变体，并在COS细胞中表达。为了确定哪些特性与LGMD2A直接相关，我们检查了表达的p94点突变体是否具有上述所列的独特特性。与连接蛋白的结合和快速自溶与LGMD2A没有直接关系，但对fodrin， calpastatin， hsp60的蛋白水解活性导致LGMD2A。因此，下一个重要的问题是确定p94的底物，该底物直接或最终激活肌肉蛋白的降解，并阐明激活肌肉蛋白降解的机制。这方面的研究正在进行中。

Calpain is a typical intracellular Ca-dependent protease and hydrolyzes various cellular proteins in a limited manner, changes their properties, and known as a biomodulator. p94 is a calpain homologue expressed specifically in skeletal muscle. p94 is unique in having various properties distinct from ordinary ubiquitous calpains, e.g. autolyzes very rapidly apparently in the absence of Ca2+, binds to connectin in skeletal muscle at least at two specific sites, contains a nuclear translocation signal, has three insertion sequences, hydrolyzes fodrin, calpastatin, HSP6O, etc.Quite recently, p94 has been identified as a gene responsible for limb-girdle type 2A muscular dystrophy (LGMD2A). We selected 10 point mutants from various mutants found in LGMD2A patients and the point mutants were expressed in COS cells. Expressed p94 point mutants were examined for the unique properties listed above in order to identify which properties are directly correlated to LGMD2A.Binding to connectin and rapid autolysis showed no direct correlation to LGMD2A but a toss of proteolytic activity against fodrin, calpastatin, HSP6O resulted in LGMD2A Thus the next important issue is to identify a substrate(s) of p94 which directly or eventually activates degradation of muscle proteins and to clarify a mechanism for activation of muscle protein degradation. Studies are now in progress along this line.