High‐Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method

Abstract Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The dysfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubiquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high‐throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.