Citation Export
DC Field | Value | Language |
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dc.contributor.author | Park, Seong Wook | - |
dc.contributor.author | Lee, Da Som | - |
dc.contributor.author | Kim, Yong Sung | - |
dc.date.issued | 2022-07-05 | - |
dc.identifier.issn | 1090-2104 | - |
dc.identifier.uri | https://aurora.ajou.ac.kr/handle/2018.oak/32671 | - |
dc.identifier.uri | https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85129300519&origin=inward | - |
dc.description.abstract | Ubiquitination is the covalent attachment of ubiquitin (Ub) to substrate proteins and regulates several cellular processes, including protein degradation. Ub ligases (E3s) bring a Ub-conjugated enzyme E2 (E2–Ub) and the target protein closer to enable ubiquitination. In this study, we engineered a U-box domain of human U-box-type E3 E4B (E4BU) to enhance its function as a Ub ligase by accelerating the rate of Ub transfer directly from Ub-loaded human E2 UbcH5b (E2(UbcH5b)–Ub) to the proximal substrate. We developed a functional screening system for the E4BU library using a yeast surface display system combined with fluorescence-activated cell sorting (FACS) to isolate functionally improved variants. This phenotypic screening system yielded an E4BU variant, E4BU(#8), which exhibited an approximately 4-fold greater Ub ligase activity rate in the yeast displayed form than that of the E4BU wild-type. When E4BU(#8) was fused to a green fluorescent protein (GFP)-specific nanobody, the fusion protein polyubiquitinated GFP in proportion to the concentration and incubation time, with an approximately 3-fold faster Ub ligase activity rate than the previously isolated E4BU(NT) variant. Importantly, the engineered E4BU(#8) retained endogenous Lys48-linked polyubiquitination activity, which is essential for substrate degradation by the 26S proteasome. Our results indicated that E4BU(#8), which binds to and allosterically stimulates E2(UbcH5b)–Ub to enhance Ub transferase activity to a substrate, may be valuable in designing biological molecules for targeted protein degradation. | - |
dc.description.sponsorship | This work was supported by the National Research Foundation of Korea (NRF) [grant number 2021R1A2C2003362 to YSK] and the Priority Research Centers Program [grant number 2019R1A6A1A11051471 to YSK)], which is funded by the Korean government ( MSIT ). The sponsors had no role in the study design; collection, analysis, and interpretation of the data; writing of the report; and decision to submit the manuscript for publication. | - |
dc.language.iso | eng | - |
dc.publisher | Elsevier B.V. | - |
dc.subject.mesh | Humans | - |
dc.subject.mesh | Saccharomyces cerevisiae | - |
dc.subject.mesh | Ubiquitin | - |
dc.subject.mesh | Ubiquitin-Conjugating Enzymes | - |
dc.subject.mesh | Ubiquitin-Protein Ligases | - |
dc.subject.mesh | Ubiquitination | - |
dc.title | Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity | - |
dc.type | Article | - |
dc.citation.endPage | 153 | - |
dc.citation.startPage | 147 | - |
dc.citation.title | Biochemical and Biophysical Research Communications | - |
dc.citation.volume | 612 | - |
dc.identifier.bibliographicCitation | Biochemical and Biophysical Research Communications, Vol.612, pp.147-153 | - |
dc.identifier.doi | 2-s2.0-85129300519 | - |
dc.identifier.pmid | 35525199 | - |
dc.identifier.scopusid | 2-s2.0-85129300519 | - |
dc.identifier.url | http://www.sciencedirect.com/science/journal/0006291X | - |
dc.subject.keyword | E3 ligase E4B | - |
dc.subject.keyword | Functional screening system | - |
dc.subject.keyword | U-box | - |
dc.subject.keyword | Ubiquitination | - |
dc.subject.keyword | Yeast surface display | - |
dc.type.other | Article | - |
dc.identifier.pissn | 0006-291X | - |
dc.description.isoa | false | - |
dc.subject.subarea | Biophysics | - |
dc.subject.subarea | Biochemistry | - |
dc.subject.subarea | Molecular Biology | - |
dc.subject.subarea | Cell Biology | - |
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