A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem...

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein turnover is regulated by ubiquitination, a posttranslational modification.
• Dysregulation of the ubiquitin proteasome system (UPS) is linked to cancer and neurodegenerative diseases.
• Targeting the UPS is a promising therapeutic strategy, necessitating advanced research tools.

Purpose of the Study:

• To develop a novel high-throughput live-cell assay for studying substrate protein ubiquitination.
• To overcome limitations of conventional ubiquitination assays, such as high protein requirements and reliance on modified ubiquitin.
• To enable efficient drug discovery for the UPS by facilitating the study of ubiquitination kinetics.

Main Methods:

• Development of a live-cell assay combining NanoBiT luminescence technology with tandem ubiquitin binding entities (TUBEs).
• Utilized the assay to study compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein.
• Characterized compounds based on their GSPT1 ubiquitination activity.

Main Results:

• Successfully demonstrated a high-throughput live-cell assay for resolving substrate ubiquitination.
• Characterized the ubiquitination activity of various compounds on GSPT1.
• Showcased the assay's portability for studying diverse target proteins.

Conclusions:

• The developed NanoBiT-TUBE assay provides a robust live-cell method for assessing substrate ubiquitination.
• This approach facilitates the study of ubiquitin transfer kinetics and compound screening.
• The assay is adaptable for investigating ubiquitination of various proteins, supporting UPS-related drug discovery.