Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Deubiquitinases (DUBs) are crucial drug targets for disease pathway modulation.
• Identifying direct DUB substrates is essential for understanding their cellular roles.
• Existing proteomic methods often capture indirect ubiquitination events.

Purpose of the Study:

• To develop a novel workflow for enriching direct DUB substrates.
• To apply this method to identify substrates of ubiquitin-specific protease 30 (USP30).

Main Methods:

• Developed a proximal-ubiquitome workflow combining APEX2 proximity labeling and K-ε-GG ubiquitination enrichment.
• Applied the workflow to study USP30 inhibition.
• Utilized mass spectrometry for identifying ubiquitination events.

Main Results:

• Identified known USP30-associated ubiquitination events on TOMM20 and FKBP8.
• Discovered LETM1 as a novel candidate substrate deubiquitinated by USP30.
• Demonstrated USP30-dependent deubiquitination of LETM1.

Conclusions:

• The proximal-ubiquitome workflow effectively enriches for direct DUB substrates.
• LETM1 is a novel USP30 substrate, highlighting its role in USP30-mediated deubiquitination.
• This approach advances the study of DUBs and their therapeutic potential.