2020 Fiscal Year Annual Research Report
Chemical Biology Studies of Ubiquitin Transfer with Synthetic Proteins
| Project/Area Number |
17H01211
|
|---|
| Research Institution | Nagoya University |
|---|
Principal Investigator |
Bode Jeffrey 名古屋大学, トランスフォーマティブ生命分子研究所, 客員教授 (90727900)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
望田 啓子 (桑田啓子) 名古屋大学, トランスフォーマティブ生命分子研究所, 特任講師 (70624352)
|
|---|
| Project Period (FY) |
2017-04-01 – 2022-03-31
|
| Keywords | タンパク質化学 / 有機合成化学 / プロテオミクス解析 / ユビキチン化 / ケミカルバイオロジー |
|---|
| Outline of Annual Research Achievements |
During this funding period (FY 2021), we attempted to trap the unknown E3 ligases for a key protein CRY1, which is involved in regulating the circadian rhythms. Among synthetic variants of E2 conjugating enzymes, we identified the suitable variant for biochemical experiments using cell lysates. The synthetic probes were prepared using the protocols that we established in previous years. By employing our synthetic E2-SUMO probes in cells and cell lysates for photoinduced trapping of candidates E3 ligases. By working with our collaborator Dr. Kuwata, we could identify several candidate E3 ligases responsible for modifications of CRY1. Based on these hits, we will continue work with another collaborator, Dr. Tsuyoshi Hirota, to validate these finding using siRNA knock downs of the candidate genes.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
sValidation of identified E3 proteins is ongoing. We examined phenotypes of the circadian rhythms by knockdown of the candidate proteins in collaboration with Dr. Hirota at ITbM. We are now trying to expand our efforts to other modifier proteins such as ISG15 by working on the chemical synthesis of the corresponding E2 enzymes. We plan to use the same approach established by our synthesis of Ubc9 to prepare specific probes of the ISG15ylation pathway.
|
| Strategy for Future Research Activity |
We will use our established probes to identify the E3 ligases responsible for SUMOylation. In addition to our ongoing work with CRY1, we will extend this to plant derived system as SUMOylation is very common in such system. It is not easy to work with plant cell lysates, however, and we will try to find suitable methods. We will also continue to prepare new E2 conjugating enzymes. We are also working on the preparation of Ubiquitin and Ubiquitin-like proteins that couple irreversibly with E2 conjugating enzymes, allowing them to be pulled down and isolated. These semi-synthetic systems, if successful, could be used to identify the unknown E2 conjugating enzymes responsible for the attachment of Ub-like modifiers including ISG15, UFM1, and NEDD8.
|
