Ubiquitin is a small protein tag that modifies proteins to regulate diverse cellular processes in eukaryotic cells. When the protein is marked with ubiquitin, it changes the function of the protein such as localization, protein-protein interaction, half-life and activity. The best known consequence is ubiquitin-mediated proteolysis, where the ubiquitin-tagged protein is targeted to the proteasome for degradation. This is an important process as it serves to maintain the cellular protein levels and to remove unwanted proteins to keep the cells healthy. Ubiquitin modification involves sequential action of three key enzymes: ubiquitin-activating enzyme, ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). E3s catalyze the final step of this reaction. They recruit E2 conjugated with ubiquitin and protein substrate to facilitate the transfer of ubiquitin from E2 to the substrate. There are 600 members in humans making them one of the largest family of proteins in cells. Over the past 15 years, various studies have paved the way for understanding how E3s interact with E2s and substrates, and how they are regulated. However, their mechanisms of ubiquitin transfer remain elusive. Understanding how E3s catalyze ubiquitin transfer is important as many of them are involved in various diseases including cancer. Furthermore several studies have shown that targeting the ubiquitin-proteasome system is a valid approach to treat diseases, for example, Velcade, a proteasome inhibitor that has been approved for treating patients with multiple myeloma. More recently, proteolysis targeting chimera, a bifunctional molecule that links E3 and a non-natural substrate, has emerged as the novel therapeutic approach to target cellular proteins for ubiquitination and degradation. Thus, studies on E3s could benefit on future development of therapeutics. The overall objectives of RINGE3 are to characterize the reaction steps catalyzed by E3 to understand how it activates E2 conjugated with ubiquitin, how it transfers ubiquitin to substrate and how it builds ubiquitin chains.
