title: Screening E3 substrates using a live phage display library.
abstract: Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage display library as E3 substrates in in vitro screening. His-ubiquitinated phage, enriched with Ni-beads, could effectively infect E. coli for amplification. Sixteen natural potential substrates and many unnatural potential substrates of E3 MDM2 were identified through 4 independent screenings. Some substrates were identified in different independent experiments. Additionally, 10 of 12 selected candidates were ubiquitinated by MDM2 in vitro, and 3 novel substrates, DDX42, TP53RK and RPL36a were confirmed ex vivo. The whole strategy is rather simple and efficient. Non-degradation substrates can be discovered. This strategy can be extended to any E3s as long as the E3 does not ubiquitinate the empty phage.
authors.affiliation: Department of Physiology and Pathophysiology, National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing, China ; Department of Core Instrument Facility, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing, China.
authors.forename: Zhengguang
authors.initials: Z
authors.lastname: Guo
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.forename: Huihua
authors.initials: H
authors.lastname: Li
authors.forename: Youhe
authors.initials: Y
authors.lastname: Gao
mesh_terms: D002460:Cell Line; D061505:Cell Surface Display Techniques; D057809:HEK293 Cells; D006801:Humans; D019151:Peptide Library; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D019151:Peptide Library; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases