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Novel cysteine proteases involved in protein metabolism in endoplasmic reticulum of rat liver

参与大鼠肝脏内质网蛋白质代谢的新型半胱氨酸蛋白酶

基本信息

批准号：
05660139
负责人：
URADE Reiko
金额：
$ 1.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660139/
关键词：
Cysteine protease Endoplasmic reticulum Cloning cDNA Expression

项目摘要

In animal cells, secretary and membrane proteins were de novo synthesized and folded in endoplasmic reticulum. Among them, the abnormal structure proteins such as mutant, misfolded and nonassociated polypeptides are segregated from the normal proteins, retained and finally degraded in the endoplasmic reticulum (quality control) . Although many molecular elements, involved in the quality control of nascent polypeptides in the endoplasmic reticulum, have been identified, protease (s) , which acts on the degradation of abnormal proteins, is not clear. In this study, novel proteases, which are candidates for members of quality control machinery, were purified from the endoplasmic reticulum of rat liver and named ER-60 protease and ER-72 protease. These were shown to be cysteine proteases which have unusual substrate specificity and sensitivities to protease inhibitors. cDNAs of rat and human ER-60 protease were cloned and sequenced. From these cDNAs, large expression systems of recombinant proteins in E.coli were established. A crystallization of recombinant ER-60 protease for X-ray analysis was tried and some conditions, that protein crystals were formed, were found. Experimental systems established in this study might be useful tools for the determination of relationships between structure and regulation of novel cysteine proteases of endoplasmic reticulum in future.

在动物细胞中，秘书蛋白和膜蛋白是在内质网中重新合成和折叠的。其中，突变蛋白、错误折叠蛋白、非相关多肽等结构异常蛋白从正常蛋白中分离出来，在内质网中保留并最终降解（质量控制）。虽然已经确定了许多参与内质网新生多肽质量控制的分子元件，但作用于异常蛋白降解的蛋白酶尚不清楚。在本研究中，从大鼠肝脏内质网中纯化了新的蛋白酶，命名为ER-60蛋白酶和ER-72蛋白酶，它们是质量控制机制成员的候选成员。这些被证明是半胱氨酸蛋白酶，具有不寻常的底物特异性和对蛋白酶抑制剂的敏感性。克隆并测序了大鼠和人ER-60蛋白酶的cdna。利用这些cdna，在大肠杆菌中建立了重组蛋白的大表达体系。对重组ER-60蛋白酶的结晶进行了x射线分析，发现了形成蛋白晶体的一些条件。本研究建立的实验系统可能为今后确定新型内质网半胱氨酸蛋白酶的结构与调控关系提供有用的工具。