Autolytic activity of human calpain 7 is enhanced by ESCRT-III-related protein IST1 through MIT-MIM interaction
Autolytic activity of human calpain 7 is enhanced by ESCRT-III-related protein IST1 through MIT-MIM interaction
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DOI：
10.1111/j.1742-4658.2010.07822.x
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发表时间：
2010-11-01
期刊：
影响因子：
5.4
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
--
作者：
Osako, Yohei;Maemoto, Yuki;Maki, Masatoshi
钙蛋白酶7是酵母Cp 11/Rim 13和真菌PalB的哺乳动物直系同源物，是一种非典型的钙蛋白酶，缺乏五EF手结构域。以前，我们报告说，一个区域包含一个串联重复的微管相互作用和运输（MIT）结构域的钙蛋白酶7相互作用的一个子集的内体分选复合物所需的运输（ESCRT）-III相关的蛋白质，这表明参与钙蛋白酶7在ESCRT系统。虽然酵母和真菌钙蛋白酶被认为是通过特定转录因子的有限蛋白水解参与碱性适应，但钙蛋白酶7的蛋白水解活性尚未得到证实。在这项研究中，我们研究了钙蛋白酶7和一个新报道的ESCRT- III家族成员，增加钠耐受性-1（IST 1），它具有两种不同类型的MIT相互作用基序（MIM 1和MIM 2）之间的相互作用。我们发现谷胱甘肽-S-转移酶（GST）融合的钙蛋白酶7的串联MIT结构域（calpain 7 MIT）拉低HEK 293 T细胞中表达的FLAG标记的IST 1。免疫共沉淀分析与各种缺失或点突变的表位标记的钙蛋白酶7和IST 1显示，重复的MIT结构域和MIMs都需要有效的相互作用。通过用GST融合的IST 1 MIM和纯化的重组钙蛋白酶7 MIT的下拉实验证实了直接的MIT-MIM结合。此外，我们发现GST-MIM蛋白增强了纯化的Strep标记的单体绿色荧光蛋白（mGFP）融合的钙蛋白酶7（mGFP-calpain 7-Strep）的自溶。自溶几乎完全废除10毫米N-乙基马来酰亚胺，但只有部分抑制1毫米亮抑酶肽或E-64。推定的催化Cys 290取代的突变体（mGFP-钙蛋白酶7（C290 S）-Strep）没有显示出自溶活性。这些结果首次证明了人钙蛋白酶7具有蛋白水解活性，并暗示钙蛋白酶7在ESCRT系统中被激活。
Calpain 7, a mammalian ortholog of yeast Cpl1/Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESCRT- III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mm N-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7(C290S)-Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system.