| 研究課題/領域番号 |
19K07373
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| 研究機関 | 東京慈恵会医科大学 |
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研究代表者 |
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| 研究分担者 |
コンプラシャ ポーンパン 奈良県立医科大学, 医学部, 助教 (70817767)
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| 研究期間 (年度) |
2019-04-01 – 2022-03-31
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| キーワード | Transporter / Cystinuria / Structure / Amino acids / SLC7 / Kidney disease |
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| 研究実績の概要 |
P482L is a mutation on amino acid transporter b0,+AT causing Japanese-type cystinuria. b0,+AT forms a heterodimeric complex with a regulatory protein rBAT. To understand the pathogenic mechanism, the PI planned to apply multiple analytical assays. Optimization of analytical methods is well progressed.
A promising approach to unveil the P482L pathogenic mechanism is an examination of 3D structures of b0,+AT - b0,+AT and the P482L mutant along with their heterodimeric partner rBAT were successfully purified. Continuing from last year, the large-scale purified proteins have been produced. The proteins were subjected to cryo-EM structural analysis by the contribution of the research collaborator team. A big achievement is we have successfully got the high-resolution 3D structure of b0,+AT - rBAT heterodimer. The structure reveals the biogenesis of the heterodimer and suggests the higher-order formation called “super dimer”. Along with the biogenesis, the structure showed rBAT is not only a regulatory protein for the complex translocation but also an indispensable subunit for transport function. Notably, the structure also implies the significance of residue P482 for protein dimerization, which is valuable information to clarify the P482L pathogenic mechanism.
To support the structural information, the PI research team conducted Transport assay using cell-based method and Immunofluorescent staining. Several pathogenic mutants including P482L were constructed and compared to the Wild-type. The roles of each mutant are predicted based on all combined results.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
Overall, the research has exceeding progress. Taking account of the structural and functional study of another member of Heterodimeric Amino acid Transporter (HAT) family, which we have published in 2019-2020, the PI and the collaborators have applied the platform methodology to solve the structure of rBAT - b0,+AT. By using the optimized strategies, we have successfully solved the 3D structure of rBAT - b0,+AT. The structure is in high resolution and, notably, covers the residue P482, where its mutation is pathogenic residue. The structure reveals the biogenesis of the heterodimer and suggests the higher-order formation called “super dimer” (dimer of heterodimer). Along with the biogenesis, the structure showed rBAT is not only a regulatory protein for the complex translocation as previously believed but also an indispensable subunit for transport function. In the current status, we are focusing on the analysis of the structure in detail. We are utilizing structural information, docking model and biochemical analysis to explain several residues, including P482 responsible for cystinuria.
In our original plan, we have included some analytical assays which were needed to be conducted at the collaborator’s laboratories (in Osaka and USA). Due to the restriction on traveling regarding the emergency control of the COVID-19 pandemic, we have changed our research plan. We rearrange our schedule to focus on the structural analysis and related strategies since we have successfully got the high-resolution rBAT - b0,+AT structure.
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| 今後の研究の推進方策 |
The future work and strategies are designed as described in the original research plan without significant change.
Based on the original plan, the researcher team will continuously analyze the structure of rBAT - b0,+AT by emphasizing the pathogenic residues, including the role of P482L. Although we could not rearrange to conduct some experiments at Columbia University, USA, due to the COVID-19 pandemic, the current analyses on structural analysis, docking model and biochemical analysis will be performed continuedly at The Jikei University, Tokyo, where is the PI’s affiliation. After finishing all analysis, our research team plans to publish the results. Accordingly, a part of our research plan also includes the manuscript preparation.
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| 次年度使用額が生じた理由 |
A part of incurring amount is a result of limitation that the PI originally planned to use for conducting research at Columbia University, USA. Due to the restriction on oversea traveling by the pandemic COVID-19, the PI could not rearrange schedule for the trip. Instead, the PI rearrange new research strategy. The new research approach is being conducted at the Jikei University and this Incurring amount will be used to support the conductivity during this FY2021 instead.
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