Analysis of Structure and Function of Proteasome Derived from Rat Liver Microsome

大鼠肝微粒体蛋白酶体的结构与功能分析

• 批准号: 11480170
• 负责人: KOIDE Takehiko
• 金额: $ 9.47万
• 依托单位: Himeji Institute of Technology
• 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 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480170/
• 关键词: Proteasome; ERb Proteasome; 20S Proteasome; Quality Control in the Endoplasmic reticulum; ER-Associated Degradation; Rat Liver; ERbプロアテソーム; ミクロソーム; タンパク分解; 小胞体品質管理機構; 小胞体プロテアソーム; タンパク質分解; ERaプロテアソーム

This investigation was aimed to characterize structural and enzymic properties of the ERb proteasome which we have isolated, for the first time in the world, from rat liver microsome fraction as an ER membrane-bound proteasome and to determine the structural element specific in the ERb proteasome, and we clarified the followings :1. On reverse-phase HPLC, in addition to β2 and α5 of 20S proteasome, ERb was found to contain extra subunits corresponding to β2 and α5 in 20S proteasome, which we referred as to B and L.2. TWO dimension gel electrophoresis revealed that B subunit is more acidic than β2 due to phosphorylation, and L subunit was essentially the same as α5, although slight difference in mobility was observed.3. On MALDI-TOF/MS, lysylendopeptidase digest of B showed the same pattern as that of β2.4. N-terminal amino acid analysis revealed that α5 has Thr and that of L was blocked. Taken together with the results of MALDI-TOF/MS, The N-terminal sequence of L was suggested as Ac-Met-Phe-Leu-Thr-where Thr was the same as N-terminal of α5.In summary, ERb contains one each of two distinctive subunits (referred to as B and L) which is absent from 20S. B is a counterpart of β2 in 20S and distinctive from β 2 in polarity and isoelectric point L is a counterpart of a5, having 3 additional residues to N-terminal of α5 and the N-terminal residue Met was acetylated. These structural feature may explain the property of ERb to bind to ER membrane.

本研究首次从大鼠肝微粒体膜结合蛋白酶体中分离得到ERb蛋白酶体，对其结构和酶学性质进行了研究，并确定了ERb蛋白酶体中特有的结构元件：1.在反相高效液相色谱上，除20S蛋白酶体的β2和α5外，还发现了与20S蛋白酶体中的β2和α5相对应的额外亚基，我们称之为B和L.2。双向凝胶电泳法显示，B亚基由于磷酸化作用比β2更酸性，而L亚基与α5基本相同，但迁移率略有不同。在MALDITOF/MS上，B的赖氨酸内切酶的酶切图谱与β2.4相同。N端氨基酸分析表明，α5含有苏氨酸受体，而L的氨基酸序列被封闭。结合MALDITOF/MS结果，建议L的N-末端序列为Ac-Met-Phe-Leu-Thr-，其中Thr与α5的N-末端相同。B是20s的β2的对应物，与β2的极性不同，等电点L是a5的对应物，在α5的N端增加了3个残基，N端的Met残基被乙酰化。这些结构特征可能解释了Erb与ER膜结合的特性。

项目成果

小出武比古: "「新生タンパク質の品質管理機構」書名「細胞の形態形成の基本メカニズム」"株式会社金芳堂. 200 (2001)

小出武彦：《新蛋白质的质量控制机制》标题：《细胞形态发生的基本机制》Kinpodo Co., Ltd. 200 (2001)

Fuminori Tokunaga: "ER-associated degradation of misfolded N-linked glycoproteins is suppressed by inhibition of ER mannosidase I"Journal of Biological Chemistry. 275. 40757-40764 (2000)

Fuminori Tokunaga：“通过抑制 ER 甘露糖苷酶 I 来抑制与 ER 相关的错误折叠 N 连接糖蛋白的降解”《生物化学杂志》。

小出武比古: "書名 細胞の形づくり 論文標題 「新生タンパク質の品質管理機構」"株式会社金芳堂(印刷中). (2001)

小出武彦：“书名：细胞成形论文标题：“新蛋白质的质量控制机制””Kinpodo Co., Ltd.（目前正在印刷）。

小出武比古: "論文「タンパク質も品質管理されている」書名「タンパク質分解の不思議」"株式会社クバプロ. 187 (2001)

Takehiko Koide：“论文‘蛋白质也受到质量控制’。书名：‘蛋白质降解之谜’。KubaPro Co., Ltd. 187 (2001)

Hiroshi Hori: "Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes"Journal of Biochemistry. 126・4. 722-730 (1999)

Hiroshi Hori：“大鼠肝微粒体内质网中的两种 20S 蛋白酶体的分离和表征”《生物化学杂志》126・4（1999）。