Mechanism of Protein Quality Control in Endoplasmic Reticulum of Animal Cell

本项目主要研究从大鼠肝脏内质网（ER）中分离并鉴定的ER-60蛋白酶（ER-60）的结构与功能之间的关系。在1998-1999年间，进行了以下实验。ER-60含有7个半胱氨酸残基，其中4个构成了两个CGHC基序，这些基序被认为包含蛋白酶的活性中心半胱氨酸残基。通过CGHC基序c端半胱氨酸残基（Cys-60和Cys-409）突变为丙氨酸的重组人ER-60实验，这些半胱氨酸残基可能是一个活性中心半胱氨酸残基(s)。C60A/C409A修饰为丙氨酸的Cys-60和Cys-409双突变酶无活性。单突变酶C60A和C409A表现出活性。这些结果表明CGHC基序的c端半胱氨酸残基与蛋白酶活性有关。ER-60已被证明与内质网腔内的底物蛋白（溶菌酶或ApoB-100）或分子伴侣蛋白（BiP和蛋白二硫异构酶）结合。用BIAcore 2000表面等离子体共振仪分析重组人ER-60与溶菌酶或腔内分子伴侣的相互作用。ER-60与溶菌酶、BiP、蛋白二硫异构酶或钙网蛋白之间存在特异性但不强的相互作用。它们的缔合和解离动力学参数不同。结果表明，BiP与未折叠多肽或ER-60的结合特性不同。在ER中被错误折叠的ER-60 （K417 ochre）被截断后在COS-1细胞中表达，并决定了COS-1细胞重新合成后的命运。过度曝光的K417赭石以不溶性聚集体形式积累。稳定表达的K417 ochre在内质网中被一种被ALLN抑制的蛋白酶降解。

This project was planned to determine the relationship between structures and functions of ER-60 protease (ER-60) which was isolated from the endoplasmic reticulum (ER) of rat liver and characterized by the head investigator.Between 1998-1999, the experiments described below were carried out.1. ER-60 contains seven cysteine residues, four of which constitute two CGHC motifs which were assumed to include an active center cysteine residue (s) of the proteases. From the experiment with the recombinant human ER-60 with site-directed mutations of the C-terminal cysteine residues (Cys-60 and Cys-409) of the CGHC motifs to alanine, these cysteine residues were suggested to be an active center cysteine residue(s). The double-mutated enzyme with Cys-60 and Cys-409 both modified to alanine (C60A/C409A) showed no activity. The single-mutated enzymes, C60A and C409A exhibited activity. These results suggest the C-terminal cysteine residues of the CGHC motifs are responsible for the protease activity.2. ER-60 has been demonstrated to associate with substrate proteins (lysozyme or ApoB-100) or molecular chaperones (BiP and protein disulfide isomerase) in the ER lumen. The interaction of the recombinant human ER-60 and lysozyme or luminal molecular chaperones was analyzed with a surface plasmon resonance apparatus, BIAcore 2000. A specific, but not strong, interaction was observed between ER-60 and lysozyme, BiP, protein disulfide isomerase or calreticulin. Their kinetic parameters of the association and dissociation were different from each other. It was demonstrated that binding properties of BiP with unfolded polypeptide or ER-60 were different.3. The truncated ER-60 (K417 ochre), which was misfolded in the ER, was expressed in the COS-1 cell and determined the its fate after de novo synthesis. The overexposed K417 ochre was accumulated as insoluble aggregates. The stably expressed K417 ochre was degraded by a protease (s) inhibited by ALLN in the ER.