Possible regulation of the conventional calpain system by skeletal muscle-specific calpain, p94/calpain 3
Possible regulation of the conventional calpain system by skeletal muscle-specific calpain, p94/calpain 3
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DOI：
10.1074/jbc.m308789200
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发表时间：
2004-01-23
影响因子：
4.8
通讯作者：
Sorimachi, H
中科院分区：
文献类型：
--
作者：
Ono, Y;Kakinuma, K;Sorimachi, H
p94（也称为钙蛋白酶3）是骨骼肌特异性钙蛋白酶，被认为是各种细胞过程中的“调节蛋白酶”。在蛋白水平上分析p94是一个迫切的问题，因为p94蛋白酶活性的丧失会导致肢带型肌营养不良症2A型。在这项研究中，我们酶的特点，p94的一个选择性剪接的变体，p94：外显子6-15-16-（p94三角洲），其中缺乏两个p94特异性插入序列。与p94相反，由于其快速、彻底和明显不依赖于Ca 2+的自溶活性，几乎没有酶促研究，p94 Delta在COS和昆虫细胞中稳定表达。p94 Delta表现出Ca 2+依赖性的酪蛋白溶解和自溶活性，以及与常规钙蛋白酶相似的抑制剂谱。然而，钙蛋白酶抑制素不抑制p94 δ，是p94 δ的底物，这与p94的性质一致，表明p94可能是常规钙蛋白酶系统的调节剂。我们还建立了一个半定量荧光共振能量转移试验，专门使用钙蛋白酶抑制蛋白序列来测量p94活性。该方法检测COS表达的p94和p94 Delta的活性，表明其具有评估体内p94活性和诊断肢带型肌营养不良症2A型的潜力。
p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94: exons 6-15-16-(p94Delta), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca2+-independent autolytic activity, p94Delta was stably expressed in COS and insect cells. p94Delta showed Ca2+-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94Delta and is a substrate for p94Delta, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94Delta, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.