Anti-mono and polyubiquitylated conjugates monoclonal antibody, clone GL3

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases. Watanabe et al. combine two previously developed strategies to identify E3 ubiquitin ligase substrates into a method, TR-TUBE that is subsequently used to identify substrates of the Parkin and TRIM28 ligases. They identify known substrates, validating the utility of the approach, and find that TRIM28 targets Cyclin A and TFIIB for degradation.

Ubiquitin specific protease-7 (USP7) is considered an attractive target for cancer therapy by promoting degradation of the tumor suppressor p53 and negatively affecting the immune response to tumors. However, the development of selective non-covalent USP7 inhibitors has proven challenging. In this work we report the NMR characterization of a weak binder from SPR screening of an in-house fragment library which reveals that it binds to the allosteric palm site of the catalytic domain. Molecular modeling combined with H-1 NMR saturation transfer difference and NOESY experiments enabled structure-based design of additional compounds showing IC50 values in the low-micromolar range with good selectivity over the closest homolog USP47. The most potent analogue represents a promising starting point for the development of novel, selective USP7 inhibitors.