Periodic Reporting for period 4 - RINGE3 (Structural and mechanistic insights into RING E3-mediated ubiquitination)
Okres sprawozdawczy: 2019-11-01 do 2020-04-30
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.
Ubiquitin ligases (E3s) are characterized by a catalytic domain that recruits E2 conjugated ubiquitin (E2~Ub). The RING-type E3s are the largest E3 family with approximately 600 members and they harbor a RING domain that binds and activates E2~Ub for catalysis. RINGE3 focuses on expanding our understanding how RING E3s catalyze Ub transfer using both structural and biochemical approaches. Over the course of this project, we have made several discoveries that provide new insights into how RING E3s are regulated. Specifically we showed how different RING domains bind and activate E2~Ub in a similar closed conformation revealing a general principle in activation of E2~Ub for catalysis. We showed that stabilization of E2~Ub in the closed conformation can be achieved via several mechanisms for different RING E3s including dimerization, loop insertion, phosphorylation and non-covalent ubiquitin binding. Using this information, we explores the biology of inhibition of E3 activity without perturbing the substrate-binding property of an E3 MDM2 and demonstrated that targeting MDM2 E3 activity offers an alternate approach to reactivate a key tumour suppressor p53. In line with this work, we developed ubiquitin variants strategies to target the RING domain. We identified inhibitory ubiquitin variants that competes against E2~Ub binding surface of the RING domain and activator ubiquitin variants that bind a remote surface on the RING domain to stabilize E2~Ub in the closed conformation. This work demonstrated a unique strategy for targeting RING E3s to further our understanding of the biological functions of E3s. Lastly, our ongoing effort has led to development of a crosslinking strategy to capture a snapshot a RING E3 in transferring Ub and in assembling Ub chain.
I expect that the results obtained from these objectives over the course of the grant will benefit both academia and commercial (biotechnology and pharmaceutical) sectors and the general public. The academia will benefit from our dissemination of new data and methodology through publications and delivering talks in conferences. This will generate new ideas and advance research development within the scientific community. Moreover, young scientists trained in this project will have an opportunity to become the future leader in the scientific field. The commercial sector will benefit with the new knowledge on the mechanisms of ubiquitin ligases. This will generate new approaches for targeting different reaction steps of ubiquitin ligases. In the long term, new therapeutics developed through the commercial sector will benefit the general public. Furthermore, the general public will benefit from our dissemination and contribution to the global future of science, for example, new biology textbooks are being written based on the new data.