ArticlePloS one2016
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.
Article in PloS one, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 51 citations in OpenAlex.
- Phosphorylation of Runx protein controls helper CD4Nature immunology · 2026Article
- Comparative proteomic profiling of receptor kinase signaling reveals key trafficking components enforcing plant stomatal development.Science advances · 2026Article
- Exploring Novel E3 Ligases and Neosubstrates for Molecular Glue Degraders and Therapeutic Applications in Cancer.Oncology research · 2026Review
- In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID.Communications biology · 2025Article
- Comparative Proteomic Profiling of Receptor Kinase Signaling Reveals Key Trafficking Components Enforcing Plant Stomatal Development.bioRxiv : the preprint server for biology · 2025Article
- HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.The Journal of cell biology · 2024Article
- Membrane-Associated Ubiquitin Ligase RING Finger Protein 152 Orchestrates Melanogenesis via Tyrosinase Ubiquitination.Membranes · 2024Article
- Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding.Nature communications · 2023Article
- CF-PPiD technology based on cell-free protein array and proximity biotinylation enzyme for in vitro direct interactome analysis.Scientific reports · 2022Article
- Regulation of p27 (Kip1) by Ubiquitin E3 Ligase RNF6.Pharmaceutics · 2022Article
- Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.Biomolecules · 2021Article
- Chromatin Regulation through Ubiquitin and Ubiquitin-like Histone Modifications.Trends in biochemical sciences · 2021Review
- Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.The EMBO journal · 2021Article
- MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIPCommunications biology · 2021Article
- Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.Frontiers in molecular biosciences · 2021Review
- RNF6 as an Oncogene and Potential Therapeutic Target-A Review.Biotech (Basel (Switzerland)) · 2020Review
- An IMiD-induced SALL4 degron system for selective degradation of target proteins.Communications biology · 2020Article
- Structural bases of IMiD selectivity that emerges by 5-hydroxythalidomide.Nature communications · 2020Article
- A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors.Biomedicines · 2020Article
- AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.eLife · 2020Article
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Authors and funding
10 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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