2019 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, 特任助教 (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 |
This year we focused on validating the ability of synthetic E2 conjugating to trap E3 ligases. To this end, we prepared more than 10 variants of the SUMO E2 conjugating enzymes Ubc9 by chemical synthesis. But replacing the Cys93 with a non-natural amino acid (DAP), we trapped recombinant SUMO3 to form the covalent E2-SUMO complex. By introducing photoaffinity labels and biotin into the synthetic protein, we trapped RanBP2, an E3 ligase, in cell lysates. These probes are currently being used to identify the unknown SUMO E3 ligases for a key protein CRY1, which is involved in regulating the circadian rhythm.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Having established the synthesis of probes based on the SUMO E2 conjugating enzyme, we have begun to expand our synthetic efforts to other ubiquitin-like modifiers. We are particularly interested in ISG15, an important but little studied ubiquitin-like protein that contains two Ub-like domains. We have established an expression system for ISG15 and initiated the synthesis of UbcH8, one of the known E2 conjugating enzymes that operates with ISG15. 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 continue our work in two tracks. First, 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.
|
Research Products
(1 results)
