Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins

PLoS One. 2016 Jun 1;11(6):e0156718. doi: 10.1371/journal.pone.0156718. eCollection 2016.


Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

Publication types

  • Validation Study

MeSH terms

  • Animals
  • Cell-Free System
  • High-Throughput Screening Assays
  • Humans
  • Mice
  • Models, Theoretical
  • Plant Proteins / metabolism*
  • Protein Binding
  • Substrate Specificity
  • Triticum / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*


  • Plant Proteins
  • Ubiquitin-Protein Ligases

Grant support

This study is supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan (TS), the Grant-in-Aid for Scientific Research on Innovative Areas (Grant No. 25117719 to TS), the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS KAKENHI Grant No. 25290077 and 26640130 to TS, Grant No. 15K18468 to HTakahashi and 15J03774 to AU), and Program to Disseminate Tenure Tracking System (to HTakahashi). All funding or sources of support received during this specific study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.