Activity-based Proteomics of E3 Ligases

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Our health and well-being are dependent on the correct functioning of the cells in our body. It is therefore imperative that we have a thorough understanding of the biochemical processes that take place within them. It is these processes that become faulty in certain diseases therefore being armed with the knowledge of their intricacies will place us in a position where we can rationally develop new therapies that alleviate their symptoms or even cure them.A cellular regulatory mechanism that affects all processes is known as protein ubiquitination. Protein ubiquitination involves the attachment of a small protein called ubiquitin to other proteins. This modification is multipurpose but one of the most significant functions is to mark damaged or unwanted proteins for destruction. Remarkably, out of the 1000's of proteins in our cells, specific proteins can be tagged with ubiquitin. This specificity is achieved by ~600 protein enzymes known as E3 ligases. If an E3 is faulty and has reduced or elevated activity, then this can manifest itself as disease. Challenges with fully understanding protein ubiquitination arise from a lack of research technologies that enable the activities of even a single E3 in a cell to be measured. Additionally, we now have strong knowledge about which proteins are tagged with ubiquitin but our ability to identify the E3 responsible is extremely difficult and new tools are urgently needed.We have recently developed some exciting and powerful technology that allows not only a single E3s activity in a cell to be measured, but the activity of tens of E3s to be measured simultaneously. This technology will revolutionise our ability to understand the roles of E3s and protein ubiquitination in both normal and diseased cells. The first part of my research proposal will establish and optimise this technology. The second part of this proposal is to develop and optimise a second technology that will facilitate our ability to identify the E3s that are responsible for attaching ubiquitin to specific substrates.