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

在动物细胞中，分泌蛋白和膜蛋白被从头合成并在内质网中折叠。其中，突变、错误折叠和非结合多肽等异常结构蛋白从正常蛋白中分离出来，保留并最终在内质网中降解(质控)。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，但作用于异常蛋白质降解的蛋白水解酶(S)尚不清楚。在本研究中，从大鼠肝脏内质网中分离纯化了一组新的蛋白水解酶，命名为ER-60和ER-72。这些被证明是半胱氨酸蛋白酶，具有不寻常的底物专一性和对酶抑制剂的敏感性。克隆并测定了大鼠和人ER-60蛋白水解酶的基因序列。在此基础上，建立了重组蛋白在大肠杆菌中的大规模表达系统。对重组ER-60酶进行了X射线分析结晶实验，找出了形成蛋白质晶体的条件。本研究建立的实验体系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。

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.