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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - RINGE3 (Structural and mechanistic insights into RING E3-mediated ubiquitination)

Teaser

Summary

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. Thus, studies on E3s may have therapeutic potential. The overall objectives 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.

Work performed

Over this period (01/05/2015-30/10/2017), I have recruited one PhD student and three postdoc fellows to initiate the proposed project. All the new members started between 01/10/2015-01/12/2015). Each postdoc fellow has worked on the three objectives in Annex 1 Description of Action, with the PhD student also working on objective 3. Given that the three objectives are related, all members of this grant have also worked as a team to fully utilize their expertise. Objective 1 focuses on elucidating how different pairs of E2-E3 complexes activate E2 conjugated with ubiquitin for transfer. Objective 2 investigates how RING E3 facilitates the transfer ubiquitin from E2 to substrate. Objective 3 investigates how RING E3 and E2 function together to build ubiquitin chains. As layout in Annex 1 Description of Action, all objectives involved designing constructs, cloning and protein purification, complex assembly and crystallization, structural determination and biochemical validation of the structure. These are being carried out and progressing accordingly. Thus far, we have demonstrated that the nature of the amino acid at the RING E3-E2â€“Ub junction plays a key role in fine-tuning the activity of the RING E3s.

Final results

The project is still ongoing. 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.