Analysis of limited proteolytic activity of calpain-7 using non-physiological substrates in mammalian cells
Analysis of limited proteolytic activity of calpain-7 using non-physiological substrates in mammalian cells
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使用哺乳动物细胞中的非生理底物分析 calpain-7 的有限蛋白水解活性
DOI：
10.1111/febs.12243
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
前本佑樹
中科院分区：
文献类型：
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作者：
諏訪敦彦・五十嵐太郎・松山秀明;ほか;松山秀明;松山秀明;前本佑樹
CalPain-7是一种真菌非经典Calain的哺乳动物同源基因，命名为PalB，是一种细胞内的半胱氨酸蛋白酶，与运输所需的内体分选复合体(ESCRT)系统有关，在真菌的碱性适应中发挥作用。尽管我们之前发现了[Osako等人。[2010][FEBS J277,4412-4426]，目前还没有确定Calain-7的生理性或非生理性底物，需要有强有力的证据表明Calain-7具有分子间蛋白分解活性的实验有用的底物。在本研究中，我们发现，当突变体与Calain-7在HEK293T细胞中过表达时，C端截短的alg-2相互作用蛋白Alix(AlixΔC)的蛋白降解是有限的，但不是全长Alix。通过与几种ESCRT蛋白的共表达，CalPain-7对AlixΔC的切割程度得到了增强。我们研究了酪蛋白(一种包括钙蛋白酶在内的各种蛋白酶的常用底物)与Bro1结构域的融合是否赋予了作为Calain-7底物的能力，但没有观察到特定的切割。然而，当无处不在的经典钙蛋白酶的内源性抑制蛋白calastatin的结构域C1与Bro1结构域融合时，融合蛋白在Calastatin的B亚结构域(钙蛋白酶的抑制中心)的C末端边缘被切割。这些结果首次证明了Calain-7具有有限的蛋白分解活性和底物选择性。此外，所开发的蛋白降解分析系统使我们能够对Calain-7进行突变分析，这不仅揭示了N端微管相互作用和运输(MIT)结构域的重要性，而且揭示了C端C2结构域对于蛋白降解活性的重要性。
Calpain‐7 is a mammalian ortholog of a fungal non‐classical calpain named PalB, which is an intracellular cysteine protease and functions in fungal alkaline adaptation in association with the endosomal sorting complex required for transport (ESCRT) system. Despite our previous finding [Osako Yet al. (2010)FEBS J277, 4412–4426] of autolytic activity, neither physiological nor non‐physiological substrates of calpain‐7 have yet been identified, and experimentally useful substrates that show robust evidence of intermolecular proteolytic activity of calpain‐7 are required. In this study, we found limited proteolysis of C‐terminally truncated ALG‐2‐interacting protein X (ALIX; (ALIXΔC), but not full‐length ALIX, when the mutant was co‐over‐expressed with calpain‐7 in HEK293T cells and analyzed by western blotting. The extent of ALIXΔC cleavage by calpain‐7 was enhanced by co‐expression with several ESCRT proteins. We investigated whether fusion of casein, a commonly used substrate for a variety of proteases including calpains, to the Bro1 domain confers the ability to serve as a substrate of calpain‐7, but no specific cleavage was observed. However, when domain 1 of calpastatin, an endogenous inhibitory protein of ubiquitous classical calpains, was fused with the Bro1 domain, the fusion protein was cleaved at the C‐terminal border of subdomain B (an inhibitory center for calpains) of calpastatin. These results demonstrate for the first time that calpain‐7 has limited proteolytic activity and substrate preference. Moreover, the proteolytic assay system developed enabled us to perform mutational analysis of calpain‐7, which revealed the importance of not only the N‐terminal microtubule‐interacting and trafficking (MIT) domains but also the C‐terminal C2 domain‐like domains for proteolytic activity.