Skeletal muscle-specific calpain is an intracellular Na+-dependent protease.
Skeletal muscle-specific calpain is an intracellular Na+-dependent protease.
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DOI：
10.1074/jbc.m110.126946
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发表时间：
2010-07-23
期刊：
影响因子：
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通讯作者：
Sorimachi H
中科院分区：
文献类型：
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作者：
Ono Y;Ojima K;Torii F;Takaya E;Doi N;Nakagawa K;Hata S;Abe K;Sorimachi H
由于Na+/K+-ATPase使细胞内[Na+]保持在较低水平，对Na+的依赖性通常被认为是细胞外酶的一种特性。然而，我们发现p94/calain 3是钙激活的细胞内“调节器蛋白水解酶”中的一种骨骼肌特异性成员，导致肢体带状肌营养不良(“钙蛋白病变”)，在没有钙离子的情况下发生了Na+依赖而不是Cs+依赖的自溶。此外，在生理浓度下，Na+和Ca~(2+)可互补激活p94的自溶。通过阻断Na+/K+-ATPase，我们证实了p94在培养细胞内的自溶。以p94：C129S敲入(p94CS-Ki)小鼠为阴性对照，进一步证实了这一点。突变研究表明，p94分子的很大一部分参与了其依赖于Na+/Ca~(2+)的自溶，这与钙病相关突变的分散位置一致，并且该酶中保守的Ca~(2+)结合序列起到了Na+感受器的作用。以Cs+/Mg~(2+)和p94CS-Ki小鼠为阴性对照的蛋白质组学分析表明，Na+和Ca~(2+)引导p94降解不同的底物。我们提出了p94的三个作用：1)增加p94对生理[Ca~(2+)]变化的敏感性，2)调节p94的底物特异性，3)调节p94作为结构成分在肌肉细胞中的作用。最后，这是第一个细胞内依赖Na+的酶的例子。
Because intracellular [Na+] is kept low by Na+/K+-ATPase, Na+ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca2+-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na+-dependent, but not Cs+-dependent, autolysis in the absence of Ca2+. Furthermore, Na+ and Ca2+ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na+/K+-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na+/Ca2+-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca2+-binding sequence in the protease acted as a Na+ sensor. Proteomic analyses using Cs+/Mg2+ and p94CS-KI mice as negative controls revealed that Na+ and Ca2+ direct p94 to proteolyze different substrates. We propose three roles for Na+ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca2+], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na+-dependent enzyme.