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

在本研究中，我分析了内质网相关蛋白降解（ERAD）的分子机制，澄清了以下几点。已知α1-抗胰蛋白酶NHK的遗传变异可被ERAD在细胞内降解。NHK是一种糖蛋白，有三个n -连接的低聚糖。构建了缺失n -糖基化位点的突变体NHK-QQQ，并分析了其降解动力学。澄清了以下几点。(1)NHK- qqq的退化速度快于NHK。(2)NHK-QQQ被蛋白酶体降解。(3)NHK在ER和高尔基体之间循环，而NHK- qqq则保留在ER中。我们已经确定了一个TRAP（转座子相关蛋白）亚基作为一个基因，其表达在内质网应激下上调。TRAP复合体由四个亚基组成，通过与转座子的结合而存在于内质网膜中，尽管该复合体的功能尚不清楚。我们发现(1)TRAP复合体的四个亚基mRNA在内质网胁迫下同时上调。(2)这种诱导依赖于内质网应激跨膜传感蛋白Ire1和转录因子XBP1的功能。(3)通过RNA干扰（RNAi）敲除一个TRAP亚基可降低TRAP复合体的表达。(4) TRAP复合物的敲除会减弱错误折叠ERAD底物的降解动力学。因此，我们认为TRAP复合体参与了ERAD底物的识别和逆转录。

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.