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

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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.