Calpain-7 binds to CHMP1B at its second α-helical region and forms aternary complex with IST1
Calpain-7 binds to CHMP1B at its second α-helical region and forms aternary complex with IST1
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Calpain-7 在其第二个 α 螺旋区域与 CHMP1B 结合，并与 IST1 形成三元复合物
DOI：
10.1093/jb/mvr071
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发表时间：
2011
影响因子：
2.7
通讯作者：
前本佑樹(*共著論文)
中科院分区：
文献类型：
--
作者：
神田啓太郎;他6人;Keisuke Oka;Keisuke Oka;Kazuki Nagashima;Annop Klamchuen;Annop Klamchuen;Annop Klamchuen;Annop Klamchuen;長島一樹;長島一樹;長島一樹;Annop Klamchuen;岡敬祐;K.Nagashima;G.Meng;S.Rahong;A.Klamchuen;K.Oka;K.Nagashima;G.Meng;S.Rahong;A.Klamchuen;長島一樹;K.Nagashima;K.Nagashima;T.Yanagida;長島一樹;柳田剛;岡敬祐;長島一樹;長島一樹;K.Nagashima;A.Klamchuen;長島一樹;長島一樹;岡敬祐;長島一樹;岡敬祐;Kazuki Nagashima;Keisuke Oka;長島一樹;柳田剛;長島一樹;岡敬祐;柳田剛;Keisuke Oka;Kazuki Nagashima;Annop Klamchuen;Takeshi Yanagida;Masaki Kanai;Keisuke Oka;Takeshi Yanagida;Takeshi Yanagida;Masaki Kanai;Annop Klamchuen;長島一樹;Annop Klamchuen;岡敬祐;Annop Klamchuen;長島一樹;岡敬祐;柳田剛;長島一樹;柳田剛;長島一樹;岡敬祐;柳田剛;長島一樹;柳田剛;岡敬祐;長島一樹;長島一樹;岡敬祐;柳田剛;長島一樹;牧正敏(*共著論文);長島一樹;前本佑樹(*共著論文)
一些参与内体分选转运复合物（ESCRT）系统的细胞内蛋白具有微管相互作用和转运（MIT）结构域，并在其C-末端含有MIT相互作用基序（MIMs）的区域与称为带电多泡体蛋白（CHMPs）的ESCRT-III蛋白家族成员结合。虽然已经报道了两种类型的MIMs（MIM 1和MIM 2），但CHMP 1B具有MIM 1，而IST 1具有MIM 1和MIM 2。先前，我们证明CHMP 1B和IST 1直接与钙蛋白酶-7（CL 7 MIT）的MIT结构域的串联重复相互作用，并且在体外通过IST 1增强钙蛋白酶-7的自溶活性，但在HEK 293 T细胞中不通过IST 1的过表达。在这项研究中，我们检测到与CHMP 1B共表达的mGFP融合的钙蛋白酶-7的自溶增强，并观察到在HEK 293 T细胞中额外共表达IST 1的进一步激活。我们发现CL 7 MIT在体外与CHMP 1B的第二个α-螺旋区域相互作用，但不与包含MIM 1的典型C-末端区域相互作用。免疫共沉淀实验表明，CL 7 MIT和CHMP 1B之间以及CL 7 MIT和IST 1之间的相互作用在额外共表达IST 1或CHMP 1B时变得更强，表明形成calpain-7、IST 1和CHMP 1B的三元复合物。此外，亚细胞分级分离分析显示钙蛋白酶-7在膜/细胞器组分的增加，伴随这些ESCRT-III家族成员蛋白的过表达。
Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1in vitrobut not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.