Chemical tools for studying conventional and non-conventional modes of ubiquitylation

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 in a position where we can rationally develop new therapies that alleviate their symptoms of or even cure them.
A cellular regulatory mechanism that affects all processes is known as protein ubiquitylation. Protein ubiquitylation involves the attachment of a small protein called ubiquitin to other proteins. This step is carried out by enzymes known as E3. Challenges with fully understanding protein ubiquitylation 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 ubiquitylation in both normal and diseased cells.

E3 ligases (E3s) mediate the transfer of ubiquitin to specific substrates and play important roles in both health and disease. The cellular regulation and function of the vast majority of the >600 E3s remain poorly understood. The activity of E3s can be regulated by posttranslational mechanisms, including phosphorylation, and aberrant E3 activity is emerging as a hallmark of certain diseases. Development of new tools that allow the unbiased assessment of the activity state of E3s in normal and diseased cells and tissues would provide field-changing insight into the cellular roles of E3s, and may uncover novel therapeutic targets. We have developed new chemical biological tools which for the first time allow the activity of endogenous HECT and RBR E3s to be determined directly. We have validated these tools against the E3 termed Parkin that is mutated in Parkinson’s disease. Our work demonstrated that these tools can be used to garner valuable insights into the regulatory aspects of a disease-relevant E3 and that they have value as a biomarker platform for assessing disease susceptibility. We have also discovered a novel class of E3 that that was only possible through the application of our tools. E3s were previously believed to be composed of RING, HECT and RBR subtypes. We have shown that there is fourth type of E3 which we term RCR. The apparent sole member of the RCR class is a neuron-associated E3 known as MYCBP2. MYCBP2 is a positive regulator of an axon degeneration pathway and hence may be a valuable therapeutic target for addressing a range of neurological disorders. Remarkably, MYCBP2 has a unique mechanism and transfers the ubiquitin molecule onto non-lysine residues within protein substrates. Future work in our lab will involve the functional studies on MYCBP2 and non-lysine ubiquitylation in axon degeneration. We will also be applying our tools to try and understand which other poorly characterised E3s might regulate disease-relevant signalling pathways.