2020 Fiscal Year Annual Research Report
Protein quality control in the nuclear membrane by autophagy
| Project/Area Number |
20F20406
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| Research Institution | The University of Tokyo |
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| Host Researcher |
水島 昇 東京大学, 大学院医学系研究科(医学部), 教授 (10353434)
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| Foreign Research Fellow |
SOLTYSIK KAMIL 東京大学, 医学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2020-11-13 – 2023-03-31
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| Keywords | Organelle / Nuclear structure / Proteome / Protein degradation / Biomembrane |
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| Outline of Annual Research Achievements |
The final goal of this project is to find proteostatic quality control mechanisms of the nuclear membrane in mammals.
Within the first 6 months of the project (2020.11.01-2021.04.01), we have focused our work on the two main issues: 1) Optimization of the TurboID probe, that covalently attaches biotin to the nuclear envelope proteins, for mass spectrometry purposes; 2) Identification of the new transmembrane proteins of the nuclear envelope and validation of results.
Both goals met the milestones. We have successfully optimized the timing and concentration of the biotin treatment. Lysis buffer, lysis conditions, and volume of the magnetic beads were selected to ensure the most favorable capture of the biotinylated proteins. Importantly, we have paid special attention to minimize contamination by proteins from cytosolic organelles.
Using the polished protocol, we have performed a series of large-scale pulldowns, following mass spectrometry, and identified candidates, previously unknown to reside in the nuclear periphery. Next, through immunoblotting, immunofluorescence, and split GFP method we verified that one of the proteins resides in and might play a role in the degradation of the nuclear envelope proteins. Moreover, using lentiCRISPR technology we have generated knockout cells and shown that our candidate is functionally linked to the proteostasis of the nuclear membrane.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Plan for the first fiscal year of the project (2020.11.01-2021.04.01) assumed optimization of the proteomics method and trial runs of the mass spectrometry, aiming to find novel nuclear membrane proteins that, perhaps, can shed light on the problem of how nuclear envelope proteins are degraded.
The study progressed more than expected. Not only did we improve sample preparations but also identified several, previously unknown proteins of the nuclear periphery. Besides, one of the proteins has been meticulously confirmed to localize in the inner nuclear membrane thus proved to be the first known E3 ubiquitin ligase of this region.
Also, within the first fiscal year of the project, we began characterizing this protein. This exceeds originally set goals. Using lentiCRISPR technology we prepared a set of knockout cells and shown that the ligase is functionally linked to the nuclear membrane, namely ubiquitinates nuclear membrane proteins. Building on this finding, we performed differential mass spectrometry of the wild type and knockout cells and observed changes in the proteome compositions that are to be studied in the following months.
Overall, all the desired endpoints for the current stage of the study have been reached. What is more, because of the positive outcome, more work than expected has been done within the first period of the trial.
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| Strategy for Future Research Activity |
Discovery of the E3 ligase of the inner nuclear membrane marks a first step to unveil proteostasis regulation of the nuclear envelope. The second fiscal year of the project (2021.04.01 - 2022.03.31) is expected to bring new challenges.
Targets for the second fiscal year are as follows: 1) Further molecular characterization of the ligase. 2) Identification of the E2 ubiquitin-conjugating enzyme(s) that partner(s) with the E3; 3) Identification of proteins that are ubiquitinated by the E3, 4) Phenotypic consequences of the E3 loss.
Details of the planned work: 1) Transmembrane proteins often work in homooligomeric complexes. Using immunoprecipitation and blue native PAGE method we will study if the E3 forms these higher-order structures. 2) The E3 belongs to the RING-type ubiquitin ligases family that do not bind ubiquitin itself but rather transfer it using the E2-conjugating enzyme. To identify the E3 partner(s) we plan to take advantage of lentiCRISPR technology and perform a mini screening of the enzyme candidates. Shortly, knockout of the involved protein should abolish ubiquitination of the inner nuclear membrane. 3) To identify proteins ubiquitinated by the E3 we will use PTMScan. In this method, ubiquitin remnant motifs (K-ε-GG) from the wild type and the E3 knockout cells are enriched, and proteins are identified by mass spectrometry. The difference between the wild type and knockout cells will point out targets of the E3. 4) Cell phenotype will be studied using electron microscopy and a wide array of biochemical methods.
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