Activity-Based Probe Profiling of RNF12 E3 Ubiquitin Ligase Function in Tonne-Kalscheuer Syndrome

Francisco Bustos, Sunil Mathur, Carmen Espejo-Serrano, Rachel Toth, C. James Hastie, Satpal Virdee (Lead / Corresponding author), Greg M. Findlay (Lead / Corresponding author)

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Abstract

Ubiquitylation enzymes are involved in all aspects of eukaryotic biology and are frequently disrupted in disease. One example is the E3 ubiquitin ligase RNF12/RLIM, which is mutated in the developmental disorder Tønne-Kalscheuer syndrome (TOKAS). RNF12 TOKAS variants largely disrupt catalytic E3 ubiquitin ligase activity, which presents a pressing need to develop approaches to assess the impact of variants on RNF12 activity in patients. Here, we use photocrosslinking activity-based probes (photoABPs) to monitor RNF12 RING E3 ubiquitin ligase activity in normal and pathogenic contexts. We demonstrate that photoABPs undergo UV-induced labelling of RNF12 that is consistent with its RING E3 ligase activity. Furthermore, photoABPs robustly report the impact of RNF12 TOKAS variants on E3 activity, including variants within the RING domain and distal non-RING regulatory elements. Finally, we show that this technology can be rapidly deployed in human pluripotent stem cells. In summary, photoABPs are versatile tools that can directly identify disruptions to RING E3 ubiquitin ligase activity in human disease, thereby providing new insight into pathogenic mechanisms.

Original languageEnglish
Article numbere202101248
Number of pages12
JournalLife Science Alliance
Volume5
Issue number11
Early online date28 Jun 2022
DOIs
Publication statusE-pub ahead of print - 28 Jun 2022

Keywords

  • Chemical probe
  • Developmental disorder
  • E3 ubiquitin ligase
  • Stem cells
  • Ubiquitin

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