| Project/Area Number |
22K06161
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Multi-year Fund |
|---|
| Section | 一般 |
|---|
| Review Section |
Basic Section 43040:Biophysics-related
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
Walinda Erik 京都大学, 医学研究科, 助教 (80782391)
|
|---|
| Project Period (FY) |
2022-04-01 – 2025-03-31
|
| Project Status |
Granted (Fiscal Year 2023)
|
| 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)
|
|---|
| Keywords | ubiquitin / linear 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.
|
| Outline of Annual Research Achievements |
Focusing on the linear ubiquitin chain assembly complex, we elucidate how it synthesizes "head-to-tail" poly-Ub chains crucial for immune signaling and cell death regulation. Specifically, we investigate the interaction between HOIL-1L and linear poly-Ub chains, revealing the molecular determinants driving their selective binding. Through NMR and biophysical methods, we unveil the dynamic process by which the NZF domain of HOIL-1L evolves into the specific linear di-Ub-bound state while excluding other potential Ub species. Our findings highlight the role of conserved electrostatic contacts and the impact of phosphorylation at threonine-207 on linear Ub affinity. This research deepens our understanding of the Ub code and offers insights valuable for immune diseases and cancer research.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Overall, the research is progressing smoothly. However, further analysis and validation experiments are needed. While we are making large progress on understanding the kinetics of binary systems, consisiting of ubiquitin chains and their rececptors, it is tricky to dissect the competitve systems. However, we have experiments lined up that can surely elucidate this elusive mechanism.
|
| Strategy for Future Research Activity |
We will continue to dissect the kinetics of ubiquitin recognition by HOIL-1L and similar ubiquitin-binding proteins by using biophysical methods.
|
