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

在动物细胞中，分泌蛋白和膜蛋白在内质网中重新合成和折叠。其中，异常结构蛋白质如突变、错误折叠和非缔合多肽与正常蛋白质分离，保留并最终在内质网中降解（质量控制）。虽然已经鉴定了许多参与内质网中新生多肽的质量控制的分子元件，但是对异常蛋白质的降解起作用的蛋白酶尚不清楚。在这项研究中，新的蛋白酶，这是候选成员的质量控制机制，从大鼠肝内质网中纯化，命名为ER-60蛋白酶和ER-72蛋白酶。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敏感性的蛋白酶抑制剂。对大鼠和人ER-60蛋白酶的cDNA进行克隆和测序。从这些cDNA，在大肠杆菌中建立了重组蛋白的大表达系统。对重组ER-60蛋白酶进行了X-射线结晶分析，找到了蛋白晶体形成的条件，为今后研究内质网半胱氨酸蛋白酶的结构与调控关系提供了实验依据。

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.