Regulation of NF-kB signaling through competitive interactions against linear polyubiquitin
Project/Area Number |
22K06161
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Multi-year Fund |
Section | 一般 |
Review Section |
Basic Section 43040:Biophysics-related
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Research Institution | Kyoto University |
Principal Investigator |
Walinda Erik 京都大学, 医学研究科, 助教 (80782391)
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Project Period (FY) |
2022-04-01 – 2025-03-31
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Project Status |
Granted (Fiscal Year 2022)
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Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2024: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2023: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2022: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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Keywords | ubiquitin / ubiquitin binding / M1-linked chains / HOIL-1L / NZF domain / zinc finger / Ubiquitin / NF-kB signaling / Binding proteins / Chemical physics / Competitive binding |
Outline of Research at the Start |
The outline is as follows: [1] Analyze HOIL-1L, NEMO, A20 binding to linear ubiquitin in vitro. First we will conduct the binary experiments for these both proteins for various chain lengths of linear polyubiquitin. These parameters will be necessary to dissect the competitive mechanism. [2] Competitive binding to linear ubiquitin in vitro. We will couple ITC, fluorescence spectroscopy, and NMR experiments together with molecular simulations to obtain a comprehensive picture of the competition between A20, NEMO, and HOIL-1L for binding to linear ubiquitin to aid NFkB fundamental understanding.
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Outline of Annual Research Achievements |
The study has progressed smoothly and produced a range of outcomes that have been disseminated through publication. Notably, the research on ubiquitin chains using molecular dynamics simulations and NMR experiments has yielded intriguing findings that support observations made by researchers in other fields, such as X-ray crystallography. Specifically, researchers have reported diverse conformations of the same ubiquitin chain under different crystallization conditions, but all of these conformations have been found to be situated along the same phase-space trajectory as identified by molecular dynamics. As a result, we are making progress in understanding the relationship between the internal dynamics of ubiquitin chains and their subsequent binding and release from ubiquitin-binding receptors. However, we are still investigating the precise kinetics of the HOIL-1L NZF-linear ubiquitin interaction and starting to comprehend how competitive processes may contribute to polyubiquitin binding selectivity and avidity. The primary outcome of the first year has been submitted for publication. However, future aspects of the study, particularly those involving chain binders other than the HOIL-1L NZF domain, are still unpublished and will be analyzed starting this year.
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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
The research progress was moderately smooth due to competing projects conducted in parallel, particularly international collaborations that required significant analysis time. While the obtained results were intriguing and satisfactory, the involvement in other projects created some hindrances in the research timeline and productivity. Further measures could be taken to ensure better allocation of research resources and optimize the utilization of available time to maximize the research outcome.
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Strategy for Future Research Activity |
The research plan for academic year 2023 involves an analysis of the kinetics of HOIL-1L binding to polyubiquitin at the atomic scale, focusing on the contribution of the individual amino acids using NMR spectroscopy and mathematical modelling. Competitive binding assays will be conducted using A20, NEMO, and other linear polyubiquitin binding proteins. The study aims to assess the relative change in binding kinetics under these conditions, providing insights into the competitive nature of polyubiquitin binding selectivity and avidity.
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Report
(1 results)
Research Products
(6 results)