ArticleBMC biology2018
Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.
Article in BMC biology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 12 citations in OpenAlex.
- Prognostic model of ubiquitination-related genes in ovarian cancer based on transcriptomic analysis and experimental validation.Frontiers in immunology · 2025Article
- A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.Bioconjugate chemistry · 2024Article
- Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting.Analytical biochemistry · 2023Article
- Ubiquitination detection techniques.Experimental biology and medicine (Maywood, N.J.) · 2023Review
- Lessons in Organic Fluorescent Probe Discovery.Chembiochem : a European journal of chemical biology · 2021Review
- RNA pull-down confocal nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-synuclein levels.Nucleic acids research · 2021Article
- A two-step resin based approach to reveal survivin-selective fluorescent probes.RSC chemical biology · 2021Article
- Resolving the Complexity of Ubiquitin Networks.Frontiers in molecular biosciences · 2020Review
- Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes.Open biology · 2018Article
- Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.BMC biology · 2018Article
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Authors and funding
10 authors at 3 institutions in 2 countries.
Funding
Abstract
backgroundThe ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging.
resultsUPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme.
conclusionsAs a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.
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