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Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity
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dc.contributor.authorPark, Seong Wook-
dc.contributor.authorLee, Da Som-
dc.contributor.authorKim, Yong Sung-
dc.date.issued2022-07-05-
dc.identifier.issn1090-2104-
dc.identifier.urihttps://aurora.ajou.ac.kr/handle/2018.oak/32671-
dc.identifier.urihttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85129300519&origin=inward-
dc.description.abstractUbiquitination 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.sponsorshipThis 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.isoeng-
dc.publisherElsevier B.V.-
dc.subject.meshHumans-
dc.subject.meshSaccharomyces cerevisiae-
dc.subject.meshUbiquitin-
dc.subject.meshUbiquitin-Conjugating Enzymes-
dc.subject.meshUbiquitin-Protein Ligases-
dc.subject.meshUbiquitination-
dc.titleEngineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity-
dc.typeArticle-
dc.citation.endPage153-
dc.citation.startPage147-
dc.citation.titleBiochemical and Biophysical Research Communications-
dc.citation.volume612-
dc.identifier.bibliographicCitationBiochemical and Biophysical Research Communications, Vol.612, pp.147-153-
dc.identifier.doi2-s2.0-85129300519-
dc.identifier.pmid35525199-
dc.identifier.scopusid2-s2.0-85129300519-
dc.identifier.urlhttp://www.sciencedirect.com/science/journal/0006291X-
dc.subject.keywordE3 ligase E4B-
dc.subject.keywordFunctional screening system-
dc.subject.keywordU-box-
dc.subject.keywordUbiquitination-
dc.subject.keywordYeast surface display-
dc.type.otherArticle-
dc.identifier.pissn0006-291X-
dc.description.isoafalse-
dc.subject.subareaBiophysics-
dc.subject.subareaBiochemistry-
dc.subject.subareaMolecular Biology-
dc.subject.subareaCell Biology-
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