A pro-fluorescent ubiquitin-based probe to monitor cysteine-based E3 ligase activity

  • Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.
Author:David Aureliano Pérez BerrocalORCiD, Thimmalapura M. VishwanathaORCiD, Daniel Horn-GhetkoORCiD, J. Josephine BotschORCiD, Laura A. HehlORCiD, Sebastian KostrhonORCiD, Mohit MisraORCiDGND, Ivan ÐikićORCiDGND, Paul P. GeurinkORCiD, Hans van DamORCiD, Brenda A. SchulmanORCiDGND, Monique P. C. MulderORCiD
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/37272691
Parent Title (English):Angewandte Chemie / International ed.
Place of publication:Weinheim
Document Type:Article
Date of Publication (online):2023/06/05
Date of first Publication:2023/06/05
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Contributing Corporation:Gesellschaft Deutscher Chemiker
Release Date:2023/07/18
Tag:Biological Activity; Drug Discovery; E3-Ligases; Fluorescent Probes; Profiling Transthiolation Activity
Issue:online version before inclusion in an issue
Article Number:e202303319
Page Number:12
Early View: Online Version before inclusion in an issue
This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 765445 to D.P.B., the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510) and NWO (VIDI Grant VI. 213.110 to M.P.C.M.). Work in B.A.S.’s lab was funded by the ERC under the H2020 research and innovation programme (ADG-789016-Nedd8Activate), by the DFG (SCHU3196/1-1, Gottfried Wilhelm Leibniz Prize), and the Max Planck Society.
Institutes:Medizin / Medizin
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International