A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets

Commun Biol. 2020 Oct 20;3(1):592. doi: 10.1038/s42003-020-01328-y.


The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / genetics
  • Gene Knockdown Techniques
  • Humans
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / methods
  • Protein Stability
  • Proteomics / methods
  • Reproducibility of Results
  • Substrate Specificity
  • Tripartite Motif-Containing Protein 28 / genetics
  • Tripartite Motif-Containing Protein 28 / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination


  • Tripartite Motif-Containing Protein 28
  • Ubiquitin-Protein Ligases
  • parkin protein