Funcioal analysis of mouse EDEM, which eaocelerales gyocprotein ERAD

内质网中新合成的蛋白质在正确折叠或组装后被分拣到分泌途径，而错误折叠或错误组装的蛋白质保留在内质网中。这种机制被称为ER质量控制。内质网中错误折叠的蛋白质被逆转录出内质网，然后被胞浆蛋白酶体降解，这被称为ERAD(内质网相关降解)。我们克隆了一个小鼠的ERAD基因，命名为EDEM(内质网降解增强α-甘露糖苷酶样蛋白)，可以加速错误折叠糖蛋白的ERAD。在本研究中，我们明确了以下几点：1.我们以α1-抗胰蛋白酶变异体(香港)(NHK)作为ERAD的模型底物。已知内质网甘露糖苷酶I(ER Man I)对甘露糖的修剪成为糖蛋白ERAD的信号。我们已经阐明，ER Man I在人胚胎肾293细胞中的过表达促进了NHK的降解。我们还发现，ER Man I和EDEM的影响是相加的。我们的结论是，错误折叠的糖蛋白在被降解之前既被ER Man I识别，也被EDEM识别。NHK在其末端附近有一个半胱氨酸残基，因此它们有可能生成二硫键二聚体。我们已经阐明，NHK在细胞内产生同源二聚体，而EDEM的共表达抑制了二聚体的形成。二硫键结合的二聚体预计会以较低的效率逆转移出内质网。相反，EDEM不影响在内质网中正确折叠的野生型α1-抗胰蛋白酶的分泌或降解。综上所述，我们期望EDEM保持错误折叠的糖蛋白的降解能力

Newly synthesized proteins in the ER(endoplasmic reticulum) are sorted out to the secretory pathway after they fold or assemble correctly, while misfoled or misassembled proteins are retained in the ER. This mechanism is known as ER quality control. Misfolded proteins in the ER are retrotranslocated out of the ER,followed by degradation by cytosolic proteasome, which is called as ERAD(ER associated degradation). We have cloned a mouse gene, and named it as EDEM(ER degradation enhancing α-mannosidase like-protein), which accelerated the ERAD of misfolded glycoproteins.In the present study, we have clarified the followings:1. We are usingα1-antitrypsin variant null (Hong Kong) (NHK)as a model substrate for ERAD. It is known that the mannose trimming by the ER Mannosidase I(ER Man I) become the signal for glycoprotein ERAD. We have clarified that overexpression of ER Man I into human embryonic kidney 293 cells enhanced the degration of NHK. We also found that the effect of ER Man I and EDEM are additive. We conclude that the misfolded glycoproteins are recognized both by ER Man I and by EDEM before they are degraded2. NHK has one cysteine residue near its Cterminus, thus it is possible that they make disulfide-bonded dimer. We have clarified that NHK make homodimer within the cells, and that coexpression of EDEM inhibited the dimer formation. Disulfide-bonded dimer is expectedly retrotraslocated out of the ER less effciently. On the contrary, EDEM did not affect the secretion or degradation of wild type α1-antitrypsin, which folds correctly in the ER. Taken together, we expect that EDEM keep the misfolded glycoprotein degradation competent