Abstract
Ubiquitylation is a post-translational modification process crucial for eukaryotic cells. The addition of ubiquitin to substrates occurs through the action of three enzymes – ubiquitin activating, ubiquitin conjugating, and ubiquitin ligases. The dysregulation of ubiquitin conjugating enzymes has been found to be associated with various forms of cancers and diseases. UbcH5b, an E2 enzyme, has been found to have a role in the ubiquitylation of p53, a tumour suppressor. The non-covalent binding of ubiquitin to a region located at the backside of UbcH5b has been found to enhance the ubiquitylation activity of UbcH5b. Furthermore, the introduction of a Ser to Arg mutation at position 22 of UbcH5b (S22R) has been shown to prevent the non-covalent binding of ubiquitin to the backside binding region.
Ubiquitin Variants (UbV) are small proteins which resemble the ubiquitin protein found in eukaryotic cells. UbVs were originally designed to inhibit deubiquitinases but have been found to be useful inhibitors in a range of scenarios. This project explores three UbVs – UbV3, UbV6, and UbV9 – which have been identified to bind to UbcH5b away from the active site and backside region. Through ubiquitin assays, UbV3 and UbV6 were found to inhibit UbcH5b activity while UbV9 is non-inhibitory. Crystal structures indicated UbV6 binds as a dimer to WT-UbcH5b and UbV9 binds as a monomer to S22R-UbcH5b. The oligomerisation of the UbVs was further explored by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation. Each of the three UbVs were found to bind to UbcH5b with affinities in the low micromolar range.
The later portion of this project sought to determine the mechanism followed by UbV3 and UbV6 to inhibit ubiquitylation. Point mutations were introduced to UbcH5b to prevent UbV6 from binding as a dimer to the E2 enzyme. Additionally, pulldown assays were performed to determine whether UbV6 prevented UbcH5b from interacting with the RNF12 E3 ligase or the Ube1 E1 enzyme. While it was determined UbV6 did not affect the UbcH5b-RNF12 interaction, it was not fully determined whether UbV6 inhibits ubiquitylation by interfering with the interaction between UbcH5b and the Ube1 E1 enzyme.
Further investigation to understand the mechanism of inhibiting UbcH5b by UbV3 and UbV6 may be useful in the future development of a therapeutic to alleviate cancers or other diseases.