A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes
Open accessPublished version of a preprint
Abstract
While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
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PROTAC-based hijacking of SKP1, the adaptor for CRL1 (SCF) complexes, also leads to TPD.
“Pinch et al. provided further evidence of this notion by showing that PROTAC-based hijacking of SKP1, the adaptor for CRL1 (SCF) complexes, also leads to TPD. 159”
From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [159]
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- Authors
- Pinch, B. J., Buckley, D. L., Gleim, S., Brittain, S. M., Tandeske, L., D'Alessandro, P. L., Hauseman, Z. J., Lipps, J., Xu, L., Harvey, E. P., Schirle, M., Sprague, E. R., Forrester, W. C., Dovala, D., McGregor, L. M., & Thoma, C. R.
- Corresponding author
- Pinch, Benika J.
- Forrester, William C.
- Dovala, Dustin
- McGregor, Lynn M.
- Thoma, Claudio R.
- ORCID
- Venue
- Cell Chemical Biology
- Publisher
- Elsevier BV
- Published
- 2022-01
- Volume
- 29
- Issue
- 1
- Pages
- 57-66.e6
- ISSN
- 2451-9448 (ISSN-L)
- 2451-9456
- OpenAlex
- W3196768014
- Cited by
- 20
- References
- 54
- Citations by year
- 2026: 1 · 2025: 6 · 2024: 4 · 2023: 3 · 2022: 4 · 2021: 2
- FWCI
- 1.02
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 384 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Cell chemical biology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- The Novartis Repository (Novartis) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Keywords
- E3 ubiquitin ligase, targeted protein degradation, coffee, VHL, BRD4, JQ1, dasatinib, Skp1, cullin-RING ligases
- MeSH terms
- Cell Line, Female, Humans, Male, Recombinant Proteins, Ubiquitin-Protein Ligases
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