Identification of the factors mediating ubiquitination of inclusion body in neurodegenerative diseases

神经退行性疾病中包涵体泛素化介导因素的鉴定

• 批准号: 11480232
• 负责人: NAKAYAMA Kei-ichi
• 金额: $ 9.54万
• 依托单位: Medical Institute of Bioregulation, Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480232/
• 关键词: ubiquitination; inclusion body; neurodegenerative disease; proteolysis; Machado-Joseph Disease; polyglutamine disease; マシャド・ジョセフ病; ユビキチン; VCP; UFD2a; ユビキチン鎖伸長因子(E4)

It has been reported that most of inclusion bodies which are found in patients' neurons of neurodegenerative diseases are ubiquitinated. The aim of this research project is to identify the factors to mediate ubiquitination of the proteins constituting the inclusion body. As a model system, we chose Machado-Joseph disease (MJD), in which polyglutamine stretch of MJD1 protein is abnormally expanded. We found that MJD1 protein undergoes ubiquitination both in vivo and in vitro. Using the in vitro ubiquitination system as an assay for detection of ubiquitinating activity, we purified the factors required for ubiquitination of MJD1 with several column chromatography. Finally, the activity was recovered in the fraction > 1,000 kDa with gel-filtration column chromatography.We finally identified some components of the ubiquitinating enzyme complex by amino acid sequencing, and isolated the cDNA encoding the proteins. Currently we examined the biological significance and involvement of the formation of inclusion body in neurodegenerative diseases.

据报道，神经退行性疾病患者神经元中发现的包涵体大部分是泛素化的。该研究项目的目的是确定介导构成包涵体的蛋白质泛素化的因素。作为模型系统，我们选择了马查多-约瑟夫病 (MJD)，其中 MJD1 蛋白的聚谷氨酰胺链段异常扩展。我们发现 MJD1 蛋白在体内和体外都会发生泛素化。利用体外泛素化系统作为检测泛素化活性的方法，我们通过多种柱层析纯化了MJD1泛素化所需的因子。最后，通过凝胶过滤柱色谱法恢复了> 1,000 kDa的级分的活性。我们最终通过氨基酸测序鉴定了泛素化酶复合物的一些成分，并分离了编码该蛋白的cDNA。目前我们研究了包涵体形成在神经退行性疾病中的生物学意义和参与。

项目成果

Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K.-i., and Nakayama, K.: "An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of β-catenin."EMBO J.. 18. 2401-2410 (1999)

北川 M.、畠山 S.、白根 M.、松本 M.、石田 N.、服部 K.、中道 I.、菊池 A.、中山 K.-i. 和中山, K.：“F-box 蛋白 FWD1 介导泛素依赖性 β-catenin 蛋白水解。”EMBO J.. 18. 2401-2410 (1999)

Lorick,K.L.: "RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination."Proc.Natl.Acad.Sci.USA. 96. 11364-11369 (1999)

Lorick, K.L.：“环指介导泛素结合酶 (E2) 依赖性泛素化。”Proc.Natl.Acad.Sci.USA。

Miura, M., Hatakeyama, S., Hattori, K., Nakayama, K.-i.: "Structure and expression of the gene encoding mouse F-Box protein, Fwd2."Genomics. 62. 50-58 (1999)

Miura, M.、Hatakeyama, S.、Hattori, K.、Nakayama, K.-i.：“编码小鼠 F-Box 蛋白 Fwd2 的基因的结构和表达。”基因组学。

Kitagawa,M.: "An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of β-catenin."EMBO J.. 8. 2401-2410 (1999)

Kitakawa, M.：“F-box 蛋白 FWD1 介导 β-catenin 的泛素依赖性蛋白水解。” EMBO J.. 8. 2401-2410 (1999)

Shimoda,K.: "Tyk2 plays a restricted role in IFNα signaling, although it is required fo IL-12-mediated T cell function"Immunity. 13. 561-571 (2000)

Shimoda, K.：“Tyk2 在 IFNα 信号传导中发挥有限作用，尽管它是 IL-12 介导的 T 细胞功能所必需的”免疫。