ArticleChemical science2016
UbFluor: A Mechanism-Based Probe for HECT E3 Ligases.
Article in Chemical science, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
What it found
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The trial behind it
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Who cites it
12 citing papers in PubMed, 24 citations in OpenAlex.
- Chemical Tools for Probing the Ub/Ubl Conjugation Cascades.Chembiochem : a European journal of chemical biology · 2025Review
- Decoding the messaging of the ubiquitin system using chemical and protein probes.Cell chemical biology · 2021Review
- Exploration of Aberrant E3 Ligases Implicated in Alzheimer's Disease and Development of Chemical Tools to Modulate Their Function.Frontiers in cellular neuroscience · 2021Review
- The role of E3 ubiquitin ligase HECTD3 in cancer and beyond.Cellular and molecular life sciences : CMLS · 2020Review
- A High-Throughput Assay for Monitoring Ubiquitination in Real Time.Frontiers in chemistry · 2019Article
- Developing Small-Molecule Inhibitors of HECT-Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities.Chembiochem : a European journal of chemical biology · 2018Review
- The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway.Cell chemical biology · 2018Article
- Monitoring PARKIN RBR Ubiquitin Ligase Activation States with UbFluor.Current protocols in chemical biology · 2018Article
- UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.The Journal of biological chemistry · 2017Article
- High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor.Current protocols in chemical biology · 2017Article
- UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis.Current protocols in chemical biology · 2017Article
- How to Target Viral and Bacterial Effector Proteins Interfering with Ubiquitin Signaling.Current topics in microbiology and immunologyReview
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Homologous to E6AP Carboxyl Terminus E3 ubiquitin ligases (HECT, ~28 known) are genetically implicated in cancer, neurological, hypertensive, and autoimmune disorders, and are potential drug targets to treat these diseases. The major bottleneck in the field of HECT E3s is a lack of simple assays to quantify the enzymatic activity of these enzymes in the presence of small molecules. Typical assays require E1, E2, HECT E3, ubiquitin (Ub), ATP and additional reagents to detect the resulting free poly-ubiquitin chains. To address this need, we developed UbFluor, a fluorescent thioester conjugate between the C-terminus of Ub and fluorescein-thiol (Fluor-SH). UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5, producing the catalytically active Rsp5~Ub (~ indicates thioester) accompanied by release of Fluor-SH. The kinetics of this two-component reaction can be easily monitored with real-time fluorescence polarization (FP) assays. Importantly, UbFluor eliminates the need to use SDS-PAGE, ATP, E1, E2 enzymes, and extra poly-ubiquitin chain detection reagents. Although the developed system lacks ATP, E1 and E2 enzymes, we show that UbFluor can recapitulate the native ubiquitination reaction by detecting and quantifying defects in transthiolation and isopeptide ligation of Rsp5 HECT E3 alanine mutants. Based on our findings, we show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.
Identifiers
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.