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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
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Methods to Detect Small Molecule Inhibition of RING E3 Ligase Activity
Ina Rothenaigner1, Jara Kerstin Brenke1, Kenji Schorpp1
1Cell Signaling and Chemical Biology, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, Neuherberg, Germany.
Current Protocols
|April 18, 2022
Summary
This study presents two methods to detect E3 ligase activity and screen for small molecule inhibitors. These assays are crucial for exploring E3 ligases as potential drug targets in cellular processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Protein ubiquitination is a critical post-translational modification regulating cellular functions.
- E3 ligases, numbering over 600, confer specificity to ubiquitination and are underrepresented drug targets.
- Monitoring E3 ligase activity is essential for identifying inhibitory small molecules.
Purpose of the Study:
- To describe protocols for detecting E3 ligase activity.
- To enable the screening of compound libraries for small molecule modulators of E3 ligases.
- To provide methods for assessing E3 ligase function and inhibition.
Main Methods:
- Development of a high-throughput AlphaScreen assay to measure TRAF6-Ubc13 interaction.
- Establishment of a low-throughput, gel-based in vitro ubiquitination assay for K63-linked chain formation.
- Utilizing these assays to test small molecule modulation of E3 ligase activity.
Main Results:
- Successful implementation of two distinct protocols for E3 ligase activity assessment.
- Demonstration of the utility of these assays in querying compound libraries.
- Validation of methods for monitoring specific E3 ligase interactions and ubiquitination events.
Conclusions:
- The presented protocols offer valuable tools for studying E3 ligase function.
- These methods facilitate the discovery of novel small molecules targeting E3 ligases.
- The study contributes to the underrepresented field of E3 ligase-targeted drug discovery.
