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Functional analysis of stomach-specific calpain.

胃特异性钙蛋白酶的功能分析。

基本信息

批准号：
15380089
负责人：
SORIMACHI Hiroyuki
金额：
$ 10.11万
依托单位：
Tokyo Metropolitan Organization for Medical Research (2004-2005)The University of Tokyo (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Calpains are a cytosolic Ca2+-regulated cysteine protease. Calpains directly interact with intracellular proteins to modify or transform functions and/or activities of their substrates, and are thus called "modulator protease". Mammals have 14 independent genes for calpains, about half of which are expressed tissue-specifically. These tissue-specific calpain are expected to be involved in specific functions developed in the specific tissues, and, therefore, their physiological functions are of great interest. One of the tissue-specific calpain, nCL-2/-2', is predominantly expressed in the stomach, especially, in the epithelial "pit" cells. The pit cells secrete mucus to protect the stomach from acid and bacteria. Therefore, nCL-2 functions are considered to be related with mucus secretion mechanisms.To elucidate physiological functions of nCL-2, we constructed nCL-2:C105S "knock-in" mice, which express a protease inactive mutant of nCL-2 instead of wild type nCL-2 under endogenous expression controls. A missense mutation corresponding to the Cys105 to Ser was introduced in the mouse genomic DNA, and neomycin-resistance gene (neoR) flanked by loxP sequences was inserted in the vicinity of the C105S mutation. After homologously targeted mice were obtained, they were crossed with Cre-recombinase expressing transgenic mice to excise neoR, resulting in nCL-2:C105S knock-in mice.Using these knock-in mice and their tissues, various biochemical, cell-biological, and molecular biological analyses have been performed. As a result, beta-COP, which is one of the subunits of COPI complex functioning in the Golgi-to-ER retrograde membrane trafficking, has been identified as a substrate for nCL-2. Moreover, nCL-2 was shown to localize in the Golgi as in the case of beta-COP. These results strongly suggest that nCL-2 functions in the context of membrane trafficking, which is brand-new concept for mode of action of calpains.

钙蛋白酶是一种胞质Ca 2+调节的半胱氨酸蛋白酶。钙蛋白酶直接与细胞内蛋白质相互作用以修饰或转化其底物的功能和/或活性，因此被称为“调节蛋白酶”。哺乳动物有14个独立的钙蛋白酶基因，其中大约一半是组织特异性表达的。这些组织特异性钙蛋白酶被认为参与了特定组织中的特定功能，因此，它们的生理功能受到了极大的关注。组织特异性钙蛋白酶nCL-2/-2 '主要在胃中表达，尤其是在上皮“小凹”细胞中。小窝细胞分泌粘液以保护胃免受酸和细菌的侵害。为了阐明nCL-2的生理功能，我们构建了nCL-2：C105 S“敲入”小鼠，其在内源性表达控制下表达nCL-2的蛋白酶失活突变体而不是野生型nCL-2。在小鼠基因组DNA中引入对应于Cys 105至Ser的错义突变，并且在C105 S突变附近插入侧翼为loxP序列的新霉素抗性基因（neoR）。在获得同源靶向小鼠后，将它们与表达Cre重组酶的转基因小鼠杂交以切除neoR，从而产生nCL-2：C105 S敲入小鼠。因此，作为在高尔基体至ER逆行膜运输中起作用的COPI复合物的亚基之一的β-COP已被鉴定为nCL-2的底物。此外，nCL-2被证明定位在高尔基体中的β-COP的情况下。这些结果有力地表明nCL-2在膜运输的背景下发挥作用，这是钙蛋白酶作用方式的全新概念。