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The Mechanisms in activation and secretion of procathepsin L in accordance with transformation

根据转化激活和分泌组织蛋白酶L的机制

基本信息

批准号：
10670145
负责人：
ISHIDOH Kazumi
金额：
$ 2.11万
依托单位：
JUNTENDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670145/
关键词：
Lysosomes Cathepsin Legumain Processing Bafilomycin A1 バフィロマイシンA1 プロカテプシン

项目摘要

This project was started by the point of view that a large amount of procathepsin L is excreted from v-Ha-ras transformed NIH3T3 cells in which mRNA for cathepsin L increases whereas a large amount of procathepsin L is excreted from its revertant cells in which mRNA for cathepsin L although does not increase, suggesting that v-ras activated the signal transduction pathways to not only the activation of cathepsin L gene but also the change in trafficing of procathepsin i to lysosomeg for excretion. Although I tried to isolate the NIH3T3 cells stably expressing rab7, its dominant-active forms and dominant negative forms, I failed to isolate the cell lines expressing rab7, its dominant active, and dominant-negative forms. Next, I try to isolate the cells expressing of rab7, its dominant-active and dominant-negative forms under the control of doxycyclin induction systems.By using peptidyl antibodies that recognized 31kDa, 30kDa, and the mature forms of cathepsin L, respectively, it was revealed that the processing of cathepsin L in vivo was the same as that in vitro.When the cells were treated with Bafilomycin A1, the intralysosomal pH increased resulting degradation of lysosomal proteinases. After this treatment, the cells were further culture in the presence of proteinase inhibitors showing the processing of legumain, cathepsins B, D, and L were carried out by papain type cysteine proteinase except for cathepsin B.This result suggested the same processing proteinase might participate in the processing of legumain, cathepsins B, D, and L.

本课题的出发点是，从组织蛋白酶L的mRNA增加的v-Ha-ras转化的NIH 3 T3细胞中分泌大量的组织蛋白酶L原，而从组织蛋白酶L的mRNA不增加的回复突变体细胞中分泌大量的组织蛋白酶L原，提示v-ras不仅激活了组织蛋白酶L基因的信号转导通路，而且改变了组织蛋白酶原i向溶酶体转运的方式。虽然我试图分离稳定表达rab 7、其显性活性形式和显性阴性形式的NIH 3 T3细胞，但我未能分离表达rab 7、其显性活性形式和显性阴性形式的细胞系。接下来，我尝试分离在强力霉素诱导系统控制下表达rab 7、其显性活性形式和显性阴性形式的细胞。通过使用分别识别31 kDa、30 kDa和成熟形式的组织蛋白酶L的肽基抗体，揭示了组织蛋白酶L在体内和体外的加工是相同的。当细胞用巴弗洛霉素A1处理时，溶酶体内pH增加，导致溶酶体蛋白酶降解。在此处理后，在蛋白酶抑制剂存在下进一步培养细胞，显示除了组织蛋白酶B外，豆荚蛋白、组织蛋白酶B、D和L的加工由木瓜蛋白酶型半胱氨酸蛋白酶进行。该结果表明，相同的加工蛋白酶可能参与豆荚蛋白、组织蛋白酶B、D和L的加工。