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

错误折叠蛋白内质网相关降解和细胞内聚集的细胞基础研究

基本信息

批准号：
13680695
负责人：
TOKUNAGA Fuminori
金额：
$ 0.83万
依托单位：
Osaka City University (2002)Himeji Institute of Technology (2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680695/
关键词：
ERAD proteasome N-linked oligosaccharide ubiquitin ligase 核内凝集体ユビキチンアグリソーム

项目摘要

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.

内质网（ER）是一个新生蛋白质的质量控制机制，在ER中的各种错误折叠或未组装的蛋白质被靶向到泛素/蛋白酶体介导的降解后，通过Sec 61易位回胞质溶胶。我们已经证明，不仅蛋白酶体抑制剂如lactacystin和carbenzoxy-leucyl-leucyl-leucinal（LLL），而且ER甘露糖苷酶I抑制剂如kifunensine和deoxymannojirimycin，强烈抑制各种错误折叠蛋白的ERAD，这表明N-连接寡糖的加工在ERAD中起着重要作用。在这项研究中，我们与东京都立医科研究所的Yoshida博士合作，发现N-聚糖是Skpl-Cullin 1-Fbx 2-Rbx 1（SCF^<Fbx2>）泛素连接酶复合物降解的信号。F-box蛋白Fbx 2特异性地结合至连接至N-连接的高甘露糖寡糖的蛋白质，并且随后有助于N-糖基化蛋白质的泛素化。此外，缺乏形成SCF复合物所必需的F-box结构域的突变体Fbx 2 ΔF的表达可明显阻断ERAD途径的典型底物的降解。我们的研究结果表明，SCF β-<Fbx2>泛素化的N-糖基化的蛋白质从ER易位到细胞质的质量控制机制。