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Analysis of the molecular mechanism ofendoplasmic reticulum-associated protein degradation (ERAD)

内质网相关蛋白降解（ERAD）分子机制分析

基本信息

批准号：
17570161
负责人：
HOSOKAWA Nobuko
金额：
$ 2.24万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In the present study, I analyzed the molecular mechanism of endoplasmic reticulum-associated protein degradation (ERAD), and the following points were clarified.1.A genetic variant of α1-antitrypsin NHK is known to be degraded intracellularly by ERAD. NHK is a glycoprotein that has three N-linked oligosaccharides. I have constructed a mutant NHK-QQQ, which lacks N-glycosylation sites, and analyzed the kinetics of degradation. The following points have been clarified. (1)NHK-QQQ was degraded more rapidly than NHK. (2)NHK-QQQ was degraded by the proteasome. (3)NHK recycles between the ER and Golgi apparatus, whereas NHK-QQQ is retained in the ER.2.We have identified a TRAP (translocon associated protein) subunit as a gene whose expression is up-regulated by ER stress. TRAP complex is composed of four subunits, and resides in the ER membrane through association with translocon, although the function of this complex is not well understood. We found that (1) the mRNA of all the four subunits of TRAP complex was up-regulated simultaneously by ER stress. (2)This induction depends on the function of Ire1, a transmembrane sensor protein for ER stress, and XBP1, a transcription factor. (3)Knock-down of one TRAP subunit by RNA interference (RNAi) decreased the expression of TRAP complex. (4)The degradation kinetics of misfolded ERAD substrates were attenuated by knock-down of the TRAP complex. Therefore, it was suggested that TRAP complex is involved in the recognition and retrotranslocation of the ERAD substrates.

本研究分析了内质网相关蛋白降解（ERAD）的分子机制，并阐明了以下几点：1.已知α1-抗胰蛋白酶NHK的一个遗传变异体可被ERAD在细胞内降解。NHK是具有三个N-连接的寡糖的糖蛋白。我构建了一个突变体NHK-QQQ，它缺乏N-糖基化位点，并分析了降解动力学。澄清了以下几点。（1）NHK-QQQ的降解速度快于NHK。（2）NHK-QQQ被蛋白酶体降解。（3）NHK在内质网和高尔基体之间穿梭，而NHK-QQQ则保留在内质网中。2.我们鉴定了一个TRAP（translocon associated protein）亚基，它的表达受内质网应激的上调。TRAP复合物由四个亚基组成，通过与易位子结合而存在于内质网膜上，但该复合物的功能尚不清楚。结果发现：（1）内质网应激可同时上调TRAP复合物四个亚基的mRNA表达;（2）这种诱导依赖于ER应激的跨膜传感蛋白Ire 1和转录因子XBP 1的功能。（3）RNA干扰（RNAi）敲低TRAP的一个亚基后，TRAP复合物的表达降低。（4）通过敲低TRAP复合物，错误折叠的ERAD底物的降解动力学减弱。因此，这表明TRAP复合物参与ERAD底物的识别和逆向转运。