Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Activity-based protein profiling (ABPP) is crucial for enzyme research and drug discovery.
• Ubiquitin E3 ligases (E3s), particularly RING E3s, are key regulators of cellular processes and attractive drug targets.
• Existing ABPP methods lack probes for measuring RING E3 ligase activity.

Purpose of the Study:

• To develop novel activity-based probes (ABPs) for RING E3 ligase activity.
• To enable activity-dependent profiling of specific RING E3 ligases and endogenous ligase activation.

Main Methods:

• Re-engineering of ubiquitin-charged E2 conjugating enzymes to create photocrosslinking ABPs.
• Demonstration of activity-dependent profiling using cancer-associated RING E3s (RNF4, c-Cbl).
• Profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation.

Main Results:

• Successful development of photocrosslinking ABPs for RING E3 ligases.
• Activity-dependent profiling of RNF4 and c-Cbl in response to native activation signals.
• Demonstration of profiling endogenous RING E3 ligase activation upon EGF stimulation.

Conclusions:

• The novel photocrosslinking ABPs are effective tools for studying RING E3 ligase activity.
• These probes facilitate research into E3 ligase biology and the development of targeted therapeutics.
• The developed ABPs advance the field of E3 ligase modulation and drug discovery.