| 研究実績の概要 |
Over a period of two years, we have developed and optimized a proximity labeling system based on TurboID. This system was utilized to uncover novel nuclear membrane proteins via the covalent attachment of biotin to peripheral nuclear proteins. The biotinylated proteins were purified and subsequently identified using mass spectrometry. A series of biochemical and microscopic techniques further validated promising hits.
One of our top candidates, a previously uncharacterized E3-ubiquitin ligase was found to exist in a high molecular weight complex via blue native PAGE analysis. To identify the components of this complex, we pull down the E3 ligase under native conditions followed by mass spectrometry. An interactome revealed proteins involved in classic nuclear processes and suggested the E3’s role in metabolic regulation. Moreover, we confirmed that the E3 responds to the cell’s metabolic state.
Furthermore, we used ubiquitome analysis (PTM scan) to find candidate target proteins for the E3. Although numerous proteins were identified in this assay, further validation of the candidate list and replication of the result is warranted to draw a final conclusion.
This critical finding requires further investigation, and we hope that it will aid in our understanding of the role of nuclear membranes in the overall function of the cell.
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