リン酸化ユビキチンを手がかりにマイトファジーの全貌にせまる

以磷酸化泛素为线索了解线粒体自噬的全貌

• 批准号: 16F15387
• 负责人: 田中 啓二
• 金额: $ 1.47万
• 依托单位: Tokyo Metropolitan Institute of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2016
• 资助国家: 日本
• 起止时间: 2016-04-22 至 2018-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16F15387/
• 关键词: mitochondria; DJ-1; Parkinson's disease; Ubiquitin; Mitochondria; Parkinson; TAT

We started the project by producing recombinant ubiquitin chains (with or without mimetic phosphorilation at S65) containing a TAT peptide. We tested the penetration capacity by treating cells with different concentrations and incubation times. To confirm the penetration into the cell, we used immunofluorescence with or without pre-permeabilization. Unfortunately we were unable to detect the presence of TAT-Ubiquitin inside the cells. The lack of detection could be due proteasome degradation, therefore we used proteasome inhibitors, but the results were the same.While we developed our approach another group published the results using a different technical approach, therefore we moved to a different topic. At the time DJ-1, a protein related to Parkinson’s disease and mitochondrial integrity, came to our attention as a new target to study. We started the study by confirming the mitochondria matrix localization of DJ-1. This is especially interesting as mechanism to explain such a sub-cellular localization change is unknown at the moment.To understand this phenomenon, we discovered several new mitochondrial localized mutants. Analyzing the characteristics of these positions in silico suggested that mitochondria-localized DJ-1 mutants are unstably folded. This led us to identify two possible cryptic mitochondrial targeting regions (MTR), and we confirmed that this MTR can import other intrinsically-disordered protein into the mitochondrial matrix. These results describe a new mechanism that allows import of pathogenic proteins into the mitochondria matrix.

我们通过生产含有达特肽的重组泛素链（在S65处有或没有模拟磷酸化）开始了该项目。我们通过用不同浓度和孵育时间处理细胞来测试渗透能力。为了确认渗透到细胞中，我们使用了有或没有预透化的免疫荧光。不幸的是，我们无法检测到细胞内TAT-泛素的存在。缺乏检测可能是由于蛋白酶体降解，因此我们使用了蛋白酶体抑制剂，但结果是一样的。当我们开发我们的方法时，另一个小组使用不同的技术方法发表了结果，因此我们转移到了不同的主题。当时DJ-1是一种与帕金森病和线粒体完整性相关的蛋白质，作为一个新的研究目标引起了我们的注意。我们通过确认DJ-1的线粒体基质定位来开始研究。这是特别有趣的机制，以解释这种亚细胞定位的变化是未知的时刻。为了理解这一现象，我们发现了几个新的线粒体定位突变体。通过计算机模拟分析这些位置的特征表明，DJ-I定位的DJ-1突变体是不稳定折叠的。这使我们确定了两个可能的隐性线粒体靶向区域（MTR），我们证实，该MTR可以将其他内在无序的蛋白质导入线粒体基质。这些结果描述了一种新的机制，允许进口的致病蛋白质进入线粒体基质。

项目成果

都医学研 Topics 中で成果報告論文が紹介される

东京医学研究所发表结果报告论文

DJ-1 and mitochondrial localization and instability case

DJ-1和线粒体定位和不稳定案例

• 发表时间: 2017
• 作者: Matsuda Noriyuki;Kimura Mayumi;Queliconi Bruno Barros;Kojima Waka;Mishima Masaki;Takagi Kenji;Koyano Fumika;Yamano Koji;Mizushima Tsunehiro;Ito Yutaka;Tanaka Keiji;Queliconi Bruno
• 通讯作者: Queliconi Bruno