Studies on Quality Control Mechanism of Proteins in Endoplasmic Reticulum

本课题是为了确定从大鼠肝的内质网（ER）中分离的ER-60蛋白酶的结构和功能之间的关系，1997年进行了下述实验：1.ER-60蛋白酶以可溶性形式定位于ER中，但不含C-末端KDEL序列，然而，ER-60蛋白酶在其C-末端位置具有独特的四肽QEDL。为了确定C-末端QEDL序列的功能，我们在COS细胞中表达野生型和突变型大鼠ER-60蛋白酶，并通过免疫染色观察蛋白酶蛋白在细胞中的定位。在用抗大鼠ER-60 F血清进行免疫染色时，未转染的COS细胞未被染色，但瞬时表达野生型大鼠ER-60蛋白酶的COS细胞被染色，显示出典型的ER谱。QEDL序列缺失或替换的突变ER-60蛋白酶 ...更多信息 由非功能性四肽AAGL组成，与高尔基体标记蛋白共定位。与表达野生型ER-60蛋白酶的细胞的情况一样，对表达KDEL取代QEDL的突变型ER-60蛋白酶的细胞的ER进行染色。野生型和KDEL取代的突变体的ER-60蛋白酶不与高尔基体标记蛋白共定位。通过<35>用抗大鼠ER-60 F血清免疫沉淀，测定COS细胞瞬时转染子中用[^ S]甲硫氨酸和-半胱氨酸脉冲标记的大鼠重组ER-60蛋白酶向培养基中的分泌。野生型酶在5小时追踪后大部分保留在细胞中。约45%的总突变体酶，其中QEDL序列被删除或替换为AAGL序列，分泌到培养基中后，5小时的追逐。ER-60蛋白酶C端的QEDL序列可能在ER中起滞留信号的作用。2.本课题组成功地在大肠杆菌BL 21（DE 3）中表达了具有蛋白水解活性的重组人ER-60蛋白。pCMB抑制了该酶的活性，证实该重组酶为半胱氨酸蛋白酶。ER-60蛋白酶含有七个半胱氨酸残基，其中四个构成两个CGHC基序，其被认为包括蛋白酶的活性中心半胱氨酸残基。从具有CGHC基序的C-末端半胱氨酸残基（Cys-60和Cys-409）定点突变为丝氨酸的重组人ER-60蛋白酶的实验中，这些半胱氨酸残基被认为是活性中心半胱氨酸残基。Cys-57和Cys-406均被修饰为丙氨酸的双突变酶（C60 A/C409 A）显示无活性。单突变的酶C60 A和C409 A表现出活性。这些结果表明CGHC基序的C-末端半胱氨酸残基负责蛋白酶活性。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