| Budget Amount *help |
¥43,810,000 (Direct Cost: ¥33,700,000、Indirect Cost: ¥10,110,000)
Fiscal Year 2021: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
Fiscal Year 2020: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
Fiscal Year 2019: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
Fiscal Year 2018: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
Fiscal Year 2017: ¥9,490,000 (Direct Cost: ¥7,300,000、Indirect Cost: ¥2,190,000)
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| 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.
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| 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.
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