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Developmental and molecular genetical analyzes on calpain-calpastatin system at individual and cellular levels

个体和细胞水平钙蛋白酶-钙蛋白酶抑制素系统的发育和分子遗传学分析

基本信息

批准号：
04454590
负责人：
EMORI Yasufumi
金额：
$ 3.97万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454590/
关键词：
Calpain Calpastatin Protease Calcium Signal transduction Drosophila Cytoskeleton Melecula biology

项目摘要

Calpain is an intracellular cysteine protease, possibly involved in Ca^<2+>-mediated signalling and activated by enhancement of intracellular Ca^<2+> concentration.First, I have identified two Drosophila genes encoding calpains. Their mRNA expression was studied by Northern blotting analysis and in situ hybridization experiment, which revealed that the two Drosophila calpains belong to conventional calpain family showing ubiquitous distribution.Next, I prepared a specific antibody against one type of the calpains. Using this antibody, cellular and subcellular localization of the calpain molecule was investigated in the early embryo. The results showed that calpain is localized with actin-related cytoskeletal structure in the very early embryo before cellularization, and in the precleavage furrows just before cellularization. These observations indicate that the Drosophila calpain is involved in univeral cellular functions, especially in the reorganization of actin-related cytoskeleton.I also studied mode of interaction between calpain and calpastatin, an endogenous inhibitor specific for calpain, by biochemical methods, which provided useful informations for further molecular studies on in vivo functions of the Drosophila calpain-calpastatin system.

Calpain是一种细胞内半胱氨酸蛋白酶，可能参与Ca^2+介导的信号传导并通过细胞内Ca^2+浓度的增强而激活。首先，我鉴定了编码calpain的两个果蝇基因。通过Northern印迹分析和原位杂交实验研究了它们的mRNA表达，结果表明这两种果蝇钙蛋白酶属于常规钙蛋白酶家族，且分布广泛。接下来，我制备了针对其中一种钙蛋白酶的特异性抗体。使用这种抗体，研究了早期胚胎中钙蛋白酶分子的细胞和亚细胞定位。结果表明，钙蛋白酶与肌动蛋白相关的细胞骨架结构一起定位于细胞化前的极早期胚胎中，以及细胞化前的卵裂前沟中。这些观察结果表明，果蝇钙蛋白酶参与了普遍的细胞功能，特别是肌动蛋白相关细胞骨架的重组。我还通过生化方法研究了钙蛋白酶与钙蛋白酶特异性内源性抑制剂钙蛋白酶抑制素之间的相互作用模式，为果蝇体内功能的进一步分子研究提供了有用的信息。钙蛋白酶-钙蛋白酶抑制素系统。