Studies on Quality Control Mechanism of Proteins in Endoplasmic Reticulum

本项目计划确定ER-60蛋白酶的结构和功能之间的关系，该蛋白酶是从大鼠肝脏内质网（ER）中分离出来的，并由首席研究员鉴定。1997年，进行了下列实验。ER-60蛋白酶以可溶性形式定位于内质网中，但不包含c端KDEL序列，这是内质网中众所周知的保留信号。然而，ER-60蛋白酶在其c端具有独特的四肽QEDL。为了确定c端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蛋白酶，并通过免疫染色观察蛋白酶蛋白在细胞中的定位。在抗大鼠ER- 60f血清免疫染色中，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，呈现典型的ER谱。将QEDL序列缺失或替换为无功能四肽AAGL的ER-60蛋白酶突变体与高尔基标记蛋白共定位。与表达野生型ER-60蛋白酶的细胞一样，用KDEL代替QEDL表达突变型ER-60蛋白酶的细胞的内质网染色。野生型和kdel取代突变型的ER-60蛋白酶与高尔基标记蛋白没有共定位。用抗大鼠ER60F血清免疫沉淀法测定COS细胞瞬时转染物中以[^<35>S]标记的大鼠重组ER-60蛋白酶向培养基中的分泌情况。野生型酶在追逐5小时后大部分留在细胞中。大约45%的突变酶（其中QEDL序列被删除或被AAGL序列取代）在追踪5小时后被分泌到培养基中。ER-60蛋白酶c端QEDL序列可能在er - 2中起保留信号的作用。在大肠杆菌（BL21(DE3)）中成功表达了具有蛋白水解活性的重组人ER-60蛋白。pCMB对其活性有抑制作用。因此，重组酶被再次确认为半胱氨酸蛋白酶。ER-60蛋白酶含有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。从重组人ER-60蛋白酶的实验中，CGHC基序的c端半胱氨酸残基（Cys-60和Cys-409）定向突变为丝氨酸，这些半胱氨酸残基被认为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。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