Chemical Biology Studies of Ubiquitin Transfer with Synthetic Proteins

2022 Fiscal Year Final Research Report

• Project/Area Number: 17H01211
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bio-related chemistry
• 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 Final Research Achievements

We prepared Ubc9 by a convergent four-segment, three-ligation strategy using serine/threonine ligation, α-ketoacid-hydroxylamine (KAHA) ligation, and native chemical ligation. This flexible, four-segment strategy was used to prepare wild-type Ubc9 as well as variants bearing combinations of side chain modifications.
The synthetic Ubc9 variants were evaluated in an SUMOylation assay using RanGAP1 as the substrate and recombinant E1 activating enzyme. To validate our key hypothesis that appropriately placed diazirines can selectively cross-link an E3 ligase bound to our synthetic Ubc9 probes, we selected RanBP2 as a test case. We attempted to trap RanBP2 using Sp100 as a substrate protein. The identity of ternary complex SUMO1-Ubc9-RanBP2 was confirmed by tandem mass spectrometry and isolated.
By employing our synthetic E2-SUMO probes in cell lysates for trapping of candidates E3 ligases. As a result, we could identify several candidate E3 ligases responsible for modifications of CRY1.

Free Research Field

有機合成化学

Academic Significance and Societal Importance of the Research Achievements

We sought to form stable, semisynthetic Ubc9-SUMO conjugates, as these are known to preferentially interact with E3 ligases. At the current state of development, this is challenging to achieve by recombinant methods including genetic code expansion.