| Project/Area Number |
23K13855
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 37030:Chemical biology-related
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| Research Institution | Kyoto University |
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Principal Investigator |
朱 浩 京都大学, 工学研究科, 助教 (90874545)
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| Project Period (FY) |
2023-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2024: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
Fiscal Year 2023: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Keywords | tyrosinase / proximity labeling / proteomics / AMPAR / mGluR1 |
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| Outline of Research at the Start |
This research will develop a new proximity labeling (PL) method based on tyrosinase, which features non-cyototoxic and compatible with diverse enrichment tags and thus enables sequential PL for precisely tracking proteome dynamics. Specifically, the proteome around AMPAR during LTD will be analyzed.
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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