Comprehensive survey of p94/calpain 3 substrates by comparative proteornics - Possible regulation of protein synthesis by p94
Comprehensive survey of p94/calpain 3 substrates by comparative proteornics - Possible regulation of protein synthesis by p94
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DOI：
10.1002/biot.200700018
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发表时间：
2007-05-01
影响因子：
4.7
通讯作者：
Sorimachi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Ono, Yasuko;Hayashi, Chikako;Sorimachi, Hiroyuki
钙蛋白酶是一种依赖钙蛋白酶的细胞质半胱氨酸蛋白酶家族，存在于几乎所有真核生物和一些细菌中，并参与多种生物现象，包括脑功能。一些calpain底物在病理条件下被积极地蛋白水解，例如，在神经退行性过程中，fodrin被calpain蛋白水解。由于在正常的生物条件下，只有非常少量的底物被钙蛋白酶水解，因此钙蛋白酶底物的分子特性在很大程度上是未知的。在本研究中，我们使用iTRAQ （TM）标记和2d LC-MALDI分析对COS7细胞中的p94/ calpain3底物进行了广泛的调查。之所以使用p94，是因为：(i)尽管p94本身是一种骨骼肌特异性钙蛋白酶，但在脑组织中仍有几种p94剪接变异体表达；（ii）它在COS细胞中表现出Ca2+独立的活性，这使得它可以在不干扰细胞的情况下评估p94蛋白酶活性对蛋白质的影响。我们的方法揭示了几种新的p94蛋白底物，包括传统calpain的底物，蛋白质合成系统的组分和糖酵解途径的酶。结果证明了该方法对钙蛋白酶底物的敏感性和实用性。该方法与其他分析方法的结合将有助于阐明calpain家族的生物学相关性。
Calpain represents a family of Call-dependent cytosolic cysteine proteases found in almost all eukaryotes and some bacteria, and is involved in a variety of biological phenomena, including brain function. Several substrates of calpain are aggressively proteolyzed under pathological conditions, e.g., in neurodegenerating processes, fodrin is proteolyzed by calpain. Because very small amounts of substrate are proteolyzed by calpain under normal biological conditions, the molecular identities of calpain substrates are largely unknown. In this study, an extensive survey of the substrates of p94/calpain 3 in COS7 cells was executed using iTRAQ (TM) labeling and 2-D LC-MALDI analysis. p94 was used because: (i) several p94 splicing variants are expressed in brain tissue even though p94 itself is a skeletal-muscle-specific calpain, and (ii) it exhibits Ca2+-independent activity in COS cells, which makes it useful for evaluating the effects of p94 protease activity on proteins without perturbing the cells. Our approach revealed several novel protein substrates for p94, including the substrates of conventional calpains, components of the protein synthesis system, and enzymes of the glycolytic pathway. The results demonstrate the usefulness and sensitivity of this approach for mining calpain substrates. A combination of this method with other analytical methods would contribute to elucidation of the biological relevance of the calpain family.