Protein quality control in the nuclear membrane by autophagy

2021 Fiscal Year Annual Research Report

• Project/Area Number: 20F20406
• Research Institution: The University of Tokyo
• Host Researcher: 水島 昇 東京大学, 大学院医学系研究科(医学部), 教授 (10353434)
• 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
• Project Period (FY): 2020-11-13 – 2023-03-31
• Keywords: Organelle / Nuclear structure / Proteome / Protein degradation / Biomembrane

Outline of Annual Research Achievements

The final goal of this project is to find proteostatic quality control mechanisms of the nuclear membrane in mammals.
Over the first six months (phase 1) of the project (2020/11/01-2021/03/31), we have developed and optimized the TurboID probe that covalently attaches biotin to the nuclear envelope proteins for mass spectrometry purposes. Using this method, we identified a set of the new transmembrane proteins of the nuclear periphery and validated the results using a series of biochemical and microscopic approaches.
In the 2nd phase of the study (2021/04/01-2022/03/31), we have focused our work on one of the candidate proteins potentially involved in proteostasis related to the nuclear envelope. Two main objectives were achieved within this period: 1) Identification of the interacting partners; 2) elucidation of the physiological role of the protein identified in phase 1.
As for finding 1, we initially found that our candidate protein exists in the high molecular weight complex. Building on this finding, we aimed to find interacting partners that were expected to shed light on the molecular mechanism of the protein action. To this end, we pull-downed candidate protein in the native conditions following mass spectrometry which led to the identification of the complex components. As for finding 2, protein of the interest was found to be active in “condition X” which explains the physiological function of the candidate protein.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Following the development and optimization of the TurboID probe that biotinylated nuclear membrane proteins, we identified candidate proteins in a series of large-scale pulldowns/mass spectrometry experiments. This opened the 2nd phase of the project.
Our plan for the second fiscal year of the project (2021/04/01-2022/03/31) assumed molecular characterization of the proteins identified in the proteomics screenings and search for their physiological function that would explain the subcellular location. This work progressed as expected in the previous report.
By analysis of the interactome, we found components of the complexes that were not known to exist in the nuclear periphery. This work required optimization of the experiment scale, selection of the appropriate beads, and adjustment of the detergent type and its concentration. Results of the mass spectroscopy analysis were confirmed by co-immunoprecipitation and western blotting.
Proteins found in the mass spectrometry screening (interactome) and confirmed to be truly interacting (western blotting), were scrutinized against available literature. Common functions of some of them were suggestive of the physiological role in which our protein complexes might work. Using this knowledge, we began a series of chemical manipulations wired with microscopic and biochemical analysis. As a result, we found “condition X” to be a key for the protein complex activation.
Overall, general endpoints for the current stage of the study have been reached.

Strategy for Future Research Activity

The discovery of the protein complexes that seem to be involved in quality control mechanisms of the nuclear envelope in mammals is an important step to better understanding the role of this membrane for whole cells. The plan for the third phase of this project is to by building on the current findings characterize each component of complexes. We aim to further understand the condition of its activation and begin the preparation of the manuscript describing these findings.Experiments and other work that are planned during the period (2022/04.01-2022.10.31) are as follows:
1) Generation of the single and multiple gene knock-out cell lines using CRISPR/Cas9 technology of each of the components of the complexes. This is required to understand the molecular function of each subunit.
2) Omics analysis of generated knock-out cells and other functional studies will be performed to link protein complexes with cellular homeostasis. These experiments will help us understand the physiological meaning of our findings.
3) Chemical manipulation using inhibitors and activators will be done to further characterize “condition X” and understand how it is linked with activated nuclear membrane complexes.
4) In parallel with experimental work, we plan the preparation of the manuscript and/or presentation of the result during scientific conferences.