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

我们通过生产含有TAT肽的重组泛素链（在S65处有或没有模拟磷酸化）开始了这个项目。我们通过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渗透的免疫荧光。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，因此我们使用蛋白酶体抑制剂，但结果是相同的。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布了结果，因此我们转向了不同的主题。当时，与帕金森病和线粒体完整性相关的蛋白质DJ-1作为一个新的研究目标引起了我们的注意。我们从确认DJ-1的线粒体基质定位开始研究。这一点特别有趣，因为解释这种亚细胞定位变化的机制目前尚不清楚。为了理解这一现象，我们发现了几个新的线粒体局部突变体。通过计算机分析这些位置的特征表明，线粒体定位的DJ-1突变体折叠不稳定。这使我们确定了两个可能的隐性线粒体靶向区域（MTR），并证实该MTR可以将其他内在无序的蛋白质导入线粒体基质中。这些结果描述了一种允许病原蛋白进入线粒体基质的新机制。

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.