Two Kinds Of Efficient Research Ubiquitin Ligase New Strategy Of The Substrate

Ubiquitination is one of the most important protein post-translational modifications in eukaryotes. Ubiquitination is a key mechanism in regulating many biological processes not only proteasome degradation, but also endocytic trafficking, DNA repair, signal transduction and protein-protein interaction. The ubiquitination process is achieved through a multiple enzymatic cascade. In this process, Ubiquitin ligases (E3s) determine specificity to ubiquitination by recognizing target substrates. Most of their substrates that account for the biology are not known. Identification of these substrates is a major challenge. Most substrates were identified by distinct approaches in different laboratories. Better,’faster and cheaper proteome-wide methods are needed to identify E3substrate.Recently, several powerful methods, including in vitro protein microarrays, in vivo label-free or stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry and global protein stability (GPS) profiling have been employed for identification of E3substrates. In this study, we developed two novel different strategies to identify substrates of E3in a large scale.Mostly, E3s specifically recognize substrates via their protein interaction domains. In the first part of this article, we developed an integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also non-substrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PBK and BCR, were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can be potentially extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.As another part of this article, we develop another proteomic strategy using live phage display library as E3substrates in screening. Screening was performed by selecting phage clones expressing proteins or peptides which are ubiquitinated by E3. We used the MDM2to illustrate our strategy.16natural potential substrates and many unnatural potential substrates of MDM2were identified through4different screenings.10of12selected candidates were ubiquitinated by MDM2in vitro, and three novel substrates, DDX42, TP53RK and RPL36a were confirmed in vivo. We further revealed that MDM2-mediated ubiquitination of TP53RK cause its proteasome dependent degradation. Not only the substrates poly-ubiquitinated but also the ones mono-/oligo-ubiquitinated by the MDM2were discovered in our strategy. This strategy can be extended to any ubiquitin ligases (E3s) for the substrates’discovery, as long as the target E3do fit for the in vitro ubiquitination system and do not ubiquitinate the empty phage.