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离子依赖性蛋白酶，能有限地水解多种细胞蛋白，改变其性质，被称为生物调节剂。p94是在骨骼肌中特异性表达的钙蛋白酶同源物。p94的独特之处在于具有不同于普通普遍存在的钙蛋白酶的各种性质，例如，在没有Ca 2+的情况下非常迅速地明显自溶，在骨骼肌中至少在两个特定位点与连接蛋白结合，含有核转位信号，具有三个插入序列，水解胞衬蛋白、钙蛋白酶抑制蛋白、HSP 6O等。最近，p94已被鉴定为引起肢带型2A型肌营养不良症（LGMD 2A）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细胞中表达点突变体。检测表达的p94点突变体的上述独特性质，以鉴定哪些性质与LGMD 2A直接相关。与连接蛋白的结合和快速自溶显示与LGMD 2A没有直接相关性，但对胞衬蛋白，钙蛋白酶抑制蛋白，因此，下一个重要的问题是确定一个或多个底物。本发明的目的在于揭示直接或最终激活肌肉蛋白质降解的p94的表达，并阐明肌肉蛋白质降解的激活机制。沿着这条路线正在进行研究。

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.