Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome

Area of Science:

• Biochemistry and Molecular Biology
• Genetics and Genomics
• Cell Biology

Background:

• Ubiquitylation enzymes, crucial for eukaryotic biology, are often implicated in disease.
• The E3 ubiquitin ligase RNF12/RLIM is associated with the developmental disorder Tønne-Kalscheuer syndrome (TOKAS).
• RNF12 variants in TOKAS patients significantly impair its catalytic E3 ubiquitin ligase activity, necessitating methods to evaluate these disruptions.

Purpose of the Study:

• To develop and validate photocrosslinking activity-based probes (photoABPs) for monitoring RNF12 RING E3 ubiquitin ligase activity.
• To assess the utility of photoABPs in distinguishing normal RNF12 activity from pathogenic contexts.
• To investigate the impact of RNF12 TOKAS variants on E3 ligase activity using photoABPs.

Main Methods:

• Application of photocrosslinking activity-based probes (photoABPs) designed to label active E3 ubiquitin ligases.
• UV-induced labeling of RNF12 by photoABPs to confirm activity-dependent engagement.
• Testing photoABPs in the context of RNF12 variants associated with Tønne-Kalscheuer syndrome (TOKAS).
• Deployment of the photoABP technology in human pluripotent stem cells.

Main Results:

• PhotoABPs successfully labeled RNF12 in a manner consistent with its RING E3 ubiquitin ligase activity.
• The probes accurately reported the diminished E3 activity caused by RNF12 TOKAS variants, including those in the RING domain and regulatory regions.
• The photoABP technology was demonstrated to be rapidly deployable in human pluripotent stem cells.

Conclusions:

• Photocrosslinking activity-based probes (photoABPs) are effective tools for directly assessing RING E3 ubiquitin ligase activity.
• This technology can identify disruptions in RNF12 activity caused by disease-associated variants, offering insights into pathogenic mechanisms of Tønne-Kalscheuer syndrome (TOKAS).
• PhotoABPs provide a versatile method for studying E3 ligase function in both normal and disease states.