Development of the substrate identification method against budding yeast SCF complex using site-specific photocross-linking in vivo

利用体内位点特异性光交联开发针对芽殖酵母 SCF 复合物的底物识别方法

• 批准号: 23657088
• 负责人: KAMURA Takumi
• 金额: $ 2.5万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-23657088/
• 关键词: タンパク質分解; ユビキチン

Ubiquitylation and subsequent proteasomal degradation of regulatory proteins control a variety of cellular processes. The E3s are responsible for recognizing and recruiting target proteins for polyubiquitylation. Relationship between E3s and substrates are largely uncharacterized because of the technical difficulty. In this study, we perform site-specific photocross-linking in vivo to identify specific substrates of SCF complex. Hard3 forms complex with Hrd1 and functions as a E3. We identify the specific binding region of Hrd3 with Hrd1 using site-specific photocross-linking in vivo. However, in these processes, we recognize that this method is not suitable for the detection of novel binding protein, because of the lacking of the binding region information. Then we perform yeast two-hybrid screening and identify p50 as a candidate of SCFmet30 substrate. Stability of p50 is regulated by SCFmet30 in the cell cycle dependent manner. We are currently investigating the functional meaning of this degradation.

调节蛋白的泛素化和随后的蛋白酶体降解控制着各种细胞过程。E3负责识别和招募多泛素化的靶蛋白。由于技术上的困难，E3和底物之间的关系在很大程度上是未知的。在这项研究中，我们在体内进行定点光交联来鉴定SCF复合体的特定底物。Hard3与Hrd1形成复合体，发挥E3的作用。我们在体内利用定点光交联法确定了Hrd3与Hrd1的特异结合区。然而，在这些过程中，我们认识到，由于缺乏结合区域的信息，这种方法不适合检测新的结合蛋白。然后进行酵母双杂交筛选，确定p50为SCFmet30底物的候选者。SCFmet30以细胞周期依赖的方式调节p50的稳定性。我们目前正在研究这种退化的功能意义。

项目成果

Hrd1ユビキチンリガーゼ複合体の動的な会合制御は小胞体の恒常性維持に必須である

Hrd1 泛素连接酶复合物的动态调节对于维持内质网稳态至关重要

• 发表时间: 2012
• 作者: 中務邦雄;ジェフ・ブロドスキー;嘉村巧
• 通讯作者: 嘉村巧

Cullin 型E3 リガーゼの機能と制御機構

Cullin型E3连接酶的功能和控制机制

• 发表时间: 2011
• 期刊: 実験医学(増刊)「細胞内のリノベーション機構 タンパク質分解系による生体制御」
• 作者: 中務邦雄;嘉村 巧
• 通讯作者: 嘉村 巧

Snapshots of the ubiquitinated substrates and the membrane-associated E3 ligase complex during the ER-associated degradation

ER 相关降解过程中泛素化底物和膜相关 E3 连接酶复合物的快照

• 发表时间: 2011
• 作者: Oda;M.;Kanoh;Y.;Watanabe;Y.;and Masai;H.;Kunio Nakatsukasa and Takumi Kamura
• 通讯作者: Kunio Nakatsukasa and Takumi Kamura

The Role of Elongin BC-Containing Ubiquitin Ligases.

• DOI: 10.3389/fonc.2012.00010
• 发表时间: 2012
• 期刊: Frontiers in oncology
• 影响因子: 4.7
• 作者: Okumura F;Matsuzaki M;Nakatsukasa K;Kamura T
• 通讯作者: Kamura T

A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation.

停滞的转递转换络合物揭示了在ER相关降解过程中底物识别与蛋白酶体降解之间的物理联系。

• DOI: 10.1091/mbc.e12-12-0907
• 发表时间: 2013-06
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Nakatsukasa K;Brodsky JL;Kamura T
• 通讯作者: Kamura T