Photocrosslinking activity-based probes for ubiquitin RING E3 ligases
- Resource Type
- Authors
- Ronald T. Hay; Sunil Mathur; Adam J. Fletcher; Emma Branigan; Satpal Virdee
- Source
- Cell Chemical Biology
- Subject
- Models, Molecular
Phenylalanine
Ubiquitin-Protein Ligases
activity-based probes
Clinical Biochemistry
Molecular Conformation
Protein degradation
Biology
Proteomics
01 natural sciences
Biochemistry
Article
Benzophenones
PROTAC
proteomics
Ubiquitin
E3 ligases
Epidermal growth factor
ubiquitin
Drug Discovery
Humans
cancer
RING E3
Molecular Biology
Pharmacology
chemistry.chemical_classification
010405 organic chemistry
Drug discovery
RNF4
Photochemical Processes
3. Good health
0104 chemical sciences
Cell biology
Ubiquitin ligase
Cross-Linking Reagents
Enzyme
chemistry
Ubiquitin-Conjugating Enzymes
protein degradation
biology.protein
Molecular Medicine
photocrosslinking
- Language
- English
- ISSN
- 2451-9456
Summary Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes.
Highlights • Photoactivated activity-based probes developed for large class of ubiquitin E3 ligases • ABPs are compatible with divergent RING E3 activation mechanisms • Parallelized E3 profiling and detection of growth factor-induced E3 activation
Activity-based probes (ABPs) are valuable research tools for studying enzyme function. Ubiquitin E3 ligases are one of the largest enzyme families yet ABPs for this enzyme class do not exist. Mathur et al. developed photocrosslinking ABPs for RING E3s and using activity-based proteomics demonstrate activity-dependent readout of diverse E3 activation.