| 研究課題/領域番号 |
23K13855
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| 研究機関 | 京都大学 |
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研究代表者 |
朱 浩 京都大学, 工学研究科, 助教 (90874545)
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| 研究期間 (年度) |
2023-04-01 – 2025-03-31
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| キーワード | tyrosinase / proximity labeling / proteomics |
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| 研究実績の概要 |
Protein functions are tightly regulated by its localization and interaction with surrounding proteins, which are dynamically altered along a biological event. This research aims at developing a new enzyme for proximity labeling (PL) that enables sequential PL for tracking the dynamics of AMPAR-surrounding proteomes. As planned in the proposal, we have successfully developed tyrosinase-based PL in phase 1. We selected a small and simple bacterial tyrosinase (BmTyr) and firstly characterized its protein labeling properties in vitro. BmTyr enables fast protein labeling and broad amino acid modifications on proteins. We further demonstrated the subcellular-resolved PL and proteomics by BmTyr in living cells. Importantly, the BmTyr-catalyzed protein tagging features H2O2-free, low-background, and compatible with diverse probes, which potentially allows BmTyr to be developed as the first PL enzyme available for sequential PL. Lastly, we have demonstrated the applicability of BmTyr in live mouse brain for synpase-resolved PL and proteomics. The relevant results have recently been published on J. Am. Chem. Soc.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
As planned in the proposal, we have successfully developed a new proximity labeling (PL) method based on tyrosinase in phase 1. Specifically, we selected a small and simple bacterial tyrosinase (BmTyr) for this purpose. We firstly characterized the protein labeling properties of BmTyr in vitro and identified its fast labeling kinetics and relatively broad amino acid coverage on proteins. We further demonstrated that BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells, which is free of cyototoxic H2O2 and offers minimal background labeling. Beyond the scheduled research in the proposal, we have applied BmTyr in vivo to unveil the surrounding proteome of a neurotransmitter receptor in its resident synapse in a live mouse brain.
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| 今後の研究の推進方策 |
This project is moving to phase 2 and 3 for tracking the dynamic proteome surrounding AMPAR during long-term depression in cultured neurons and in live mouse brains. As proof-of-principle, sequential proximity labeling (PL) will firstly be demonstrated in HEK293T cells for organelle labeling, e.g. nucleus or endoplasmic reticulum, in which the orthogonal enrichment of labeled proteins by two distinct types of phenol probes will be tested and optimized. Afterwards, BmTyr will be anchored to AMPAR via genetic engineering or affinity binding with an appropriate ligand or nanobody. The performance of BmTyr may need get evolved, e.g., enzymatic activity, for the sophisticated sequential PL.
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| 次年度使用額が生じた理由 |
Due to the long delivery time of some oversea products, e.g., plasmids, and trials and errors on the optimization of organelle proteomics, the experiments were slightly delayed relative to the proposed timeline. The unused amounts will be applied in the next fiscal year for purchasing necessary good, e.g., antibodies and dyes, and attending international conferences.
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