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的蛋白水解酶，它以有限的方式降解各种细胞蛋白，改变其性质，是一种生物调节剂。P94是一种钙蛋白同源物，在骨骼肌中特异表达。P94具有许多不同于普通普遍存在的钙蛋白酶的特性，例如，在没有钙的情况下，自溶非常迅速，至少与骨骼肌中的连接素结合两个特定部位，包含一个核转位信号，有三个插入序列，能水解Fodrin，calastatin，HSP60等。最近，p94被发现是一个与肢体带状2A型肌营养不良症(LGMD2A)有关的基因。我们从LGMD2A患者中发现的各种突变中选择了10个点突变，并在COS细胞中表达了这些点突变。对表达的p94点突变进行了检测，以确定哪些特性与LGMD2A直接相关，与连接素的结合和快速自溶与LGMD2A没有直接关系，但对fodrin、calastatin、HSP60的蛋白分解活性的改变导致了LGMD2A。因此，下一个重要的问题是寻找p94的底物(S)，它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。目前，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.