Studies on Quality Control Mechanism of Proteins in Endoplasmic Reticulum

本项目旨在确定从大鼠肝脏内质网(ER)中分离的ER-60蛋白水解酶的结构与功能之间的关系。1997年，我们进行了以下实验：1.ER-60蛋白水解酶以可溶形式定位于内质网，但不包含已知的内质网保留信号KDEL序列，而ER-60蛋白水解酶在其C-末端有一个独特的四肽QEDL。为了确定QEDL序列的功能，我们在COS细胞中表达了野生型和突变型大鼠ER-60蛋白，并通过免疫染色观察了这些蛋白在细胞中的定位。用大鼠ER-60F抗血清免疫染色，未转染的COS细胞未见染色，而瞬时表达野生型ER-60酶的COS细胞呈典型的内质网染色。突变的ER-60蛋白水解酶，其QEDL序列缺失或复制…更多由非功能四肽AAGL与高尔基体标记蛋白共定位。用KDEL取代QEDL表达突变型ER-60酶的细胞，与表达野生型ER-60酶的细胞一样，进行ER染色。野生型和KDEL替换突变体的ER-60蛋白没有与高尔基体标记蛋白共定位。用大鼠ER60F抗血清免疫沉淀法测定瞬时转染型COS细胞中脉冲标记的大鼠重组ER-60酶的分泌量。经过5小时的追逐，野生型酶大部分仍留在细胞内。其中QEDL序列被缺失或被AAGL序列取代的突变酶中，大约45%的突变酶在5h的追逐后分泌到培养基中。2.本课题组成功地在大肠杆菌BL21(DE3)中表达了具有蛋白分解活性的重组人ER-60蛋白。重组酶的活性被pCMB抑制，证实为半胱氨酸蛋白酶。ER-60蛋白水解酶含有7个半胱氨酸残基，其中4个构成了两个CGHC基序，推测该基序中含有一个活性中心半胱氨酸残基(S)。将重组人ER-60蛋白水解酶的C端半胱氨酸残基(Cys-60和Cys-409)定点突变为丝氨酸的实验表明，这些半胱氨酸残基是一个活性中心半胱氨酸残基(S)。Cys-57和Cys-406均修饰为丙氨酸的双突变酶(C60A/C409A)无活性。单突变酶C60A和C409A表现出较强的活性。这些结果表明，CGHC基序的C端半胱氨酸残基与该酶的活性有关。较少

This project was planned to determine the relationship between structures and functions of ER-60 protease which was isolated from the endoplasmic reticulum (ER) of rat liver and characterized by the head investigator.In 1997, the experiments described below were carried out.1.ER-60 protease is localized in ER in a soluble form but does not contain a C-terminal KDEL sequence, the well-known retention signal in ER.However, ER-60 protease has the unique tetrapeptide, QEDL,at its C-terminal position. To determine the function of the C-terminal QEDL sequence, we expressed the wild-type and mutant rat ER-60 proteases in COS cells, and observed the localization of the protease proteins in the cells by immunostaining. On immunostaining with anti-rat ER-60F serum, untransfected COS cells were not stained, but the COS cells transiently expressing the wild type rat ER-60 protease were stained, showing a typical ER profile. The mutant ER-60 protease, of which the QEDL sequence was deleted or repla … More ced by a non-functional tetrapeptide, AAGL,were colocalized with the Golgi marker proteins. The ER of cells expressing the mutant ER-60 protease with KDEL substituted for QEDL was stained, as in the case of the cells epressing the wild-type ER-60 protease. The ER-60 proteases of the wild type and the KDEL-substituted mutant were not colocalized with the Golgi marker proteins. The secretion into the medium of the rat recombinant ER-60 protease pulse-labeled with [^<35>S] methinonine and-cysteine in transient transfectants of COS cells was determined by immunoprecipitation with the anti-rat ER60F serum. The wild-type enzyme mostly remained in cells after a 5-h chase. About 45% of the total mutant enzyme, of which the QEDL sequence was deleted or replaced by an AAGL sequence, was secreted into the medium after a 5-h chase. It is likely that the C-terminal QEDL sequence of ER-60 protease may function as a retention signal in ER.2.The head investigator succeeded in the expression of the recombinant human ER-60 proteas, which had the proteolytic activity, in E.coli (BL21(DE3)). The activity was inhibited by pCMB.The recombinant enzyme was thus reconfirmed to be a cysteine protease. ER-60 protease 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 proteases with site-directed mutations of the C-terminal cysteine residues (Cys-60 and Cys-409) of the CGHC motifs to serine, these cysteine residues were suggested to be an active center cysteine residue (s) . The double-mutated enzyme with Cys-57 and Cys-406 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. Less