Studies on cellular basis of the endoplasmic reticulum-associated degradation and intracellular aggregation of misfolded proteins

内质网（ER）被认为是新生蛋白的质量控制机制，内质网中各种错误折叠或未组装的蛋白在通过Sec61易位逆转录到细胞质后被泛素/蛋白酶体介导的降解所靶向。我们的研究表明，不仅蛋白酶体抑制剂如lactacystin和carbobenzoxy-leucyl-leucyl- leucyl-leucinal (LLL)，而且内质网甘露糖苷酶I抑制剂如kifunensin和deoxynannojirimycin都能强烈抑制各种错误折叠蛋白的ERAD，这表明n -连接低聚糖的加工在ERAD中起重要作用。在这项研究中，我们与东京都医学研究所的吉田博士合作，发现n -聚糖作为Skpl-Cullin1-Fbx2-Rbx1 （SCF^<Fbx2>）泛素连接酶复合物降解的信号。F-box蛋白Fbx2特异性结合到n -连接的高甘露糖寡糖上的蛋白，随后促进n -糖基化蛋白的泛素化。此外，突变体Fbx2ΔF缺乏形成SCF复合物所必需的F-box结构域，其表达明显阻碍了ERAD途径中典型底物的降解。我们的研究结果表明，SCF^<Fbx2>泛素化n -糖基化蛋白通过质量控制机制从内质网转移到细胞质。

The endoplasmic reticuium (ER) is known to function as a quality control machinery of nascent proteins, and various misfolded or unassembled proteins in the ER are targeted to ubiquitin/proteasome-mediated degradation after retrotranslocation to the cytosol through the Sec61 translocon. We have shown that not only proteasome inhibitors such as lactacystin and carbobenzoxy-leucyl-leucyl-leucinal (LLL), but also inhibitors of ER mannosidase I such as kifunensine and deoxyrnannojirimycin, strongly inhibit the ERAD of various misfolded proteins, suggesting that processing of N-linked oligosaccharide plays an important role on ERAD. In this study, collaborating with Dr. Yoshida in Tokyo Metropolitan Institute of Medical Science, we found that N-glycan serves as a signal for degradation by the Skpl-Cullin1-Fbx2-Rbx1 (SCF^<Fbx2>) ubiquitin ligase complex. The F-box protein Fbx2 binds specifically to proteins attached to N-inked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. In addition, expression of the mutant Fbx2ΔF, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ERAD pathway. Our results indicate that SCF^<Fbx2> ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.