Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system
Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system
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DOI：
10.1074/jbc.m601029200
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发表时间：
2006-07-07
影响因子：
4.8
通讯作者：
Sorimachi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Ono, Yasuko;Torii, Fukuyo;Sorimachi, Hiroyuki
p94/calpain 3 是 Ca2+ 调节的胞质半胱氨酸蛋白酶家族（钙蛋白酶）的骨骼肌特异性成员。源自基因突变的 p94 蛋白酶活性缺陷会导致称为钙蛋白酶病的肌肉营养不良症，这表明 p94 对于肌肉生存至关重要。由于 p94 特异性区域 IS1 和 IS2 的存在，p94 经历非常快速且彻底的自溶。为了阐明这种独特活性的生理相关性，研究了 p94 的自溶特征以及 p94 结合蛋白连接蛋白/肌联蛋白对此过程的影响。 p94 自溶的体外分析表明，IS1 中的自溶不会立即分解成片段，而 IS2 中新发现的隐蔽自溶位点对于分解自溶片段至关重要。作为半定量测定 p94 自溶的遗传系统，p94 在酵母中表达为酵母转录激活剂 Gal4 的 DNA 结合域和激活域之间的杂合蛋白。 Gal4-p94:WT 杂合蛋白的转录激活被 p94 自溶所阻止。通过半定量恢复 Gal4 依赖性 β-半乳糖苷酶基因表达，可以检测 C129S 活性位点突变、肢带型肌营养不良症 2A 型致病性错义突变或基于 PCR 的随机诱变导致的自溶活性完全或部分丧失。使用该系统，与 p94 结合的 N2A 连接蛋白片段被证明可以抑制 p94 自溶分解。 p94 中 IS2 自溶和连接蛋白结合位点的接近表明 N2A 连接蛋白抑制 IS2 自溶。这些数据表明 p94-连接蛋白相互作用在通过调节 p94 自溶衰变来控制 p94 功能中的重要性。
p94/calpain 3 is a skeletal muscle-specific member of the Ca2+-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94: WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.