Activity-based Proteomics of E3 Ligases

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.

I have recently developed the first activity-based probes (ABPs) that enable profiling of the transthiolation activity between E2~Ub and E3. This proposal will further develop this technology resulting in two urgently needed chemical proteomic technologies for studying the ubiquitin system. The first will be the development of a chemical proteomics platform for parallel activity-based profiling of the ~50 HECT and RBR E3 ligases found in mammalian cells. This will involve preparing new ABPs bearing biotin affinity handles. These ABPs will enable covalent labelling of active HECT/RBR E3s in cell lysates extracted from cells under investigation. Subsequent enrichment followed by quantitative mass spectrometry will enable active E3s to be identified. This will serve to identify novel therapeutic targets, as a biomarker and as a driver for further biological investigation.
Furthermore, proteomic studies have identified ~20000 ubiquitination sites within ~5000 proteins. However, there are currently no satisfactory methods that can assign an E3 to a substrate and a distinct ubiquitination site within it. The second technology I will develop will facilitate the identification of E3-substrate pairs based on both substrate identity and ubiquitination site. This will require the preparation of derivatives of my ABPs that also contain a photocrosslinking moiety. These probes will have the ability to tether RING E3 to substrate in an activity-based manner. Subsequent mass spectrometry will be used to identify the tethered E3s. A significant feature of these experimental designs is that all E3s will be endogenous protein in cell extracts. Owing to the sheer number of E3s present in humans and complications associated with their heterologous expression, this feature makes this technology particularly revolutionary and exhaustive. This proposal appears to be an excellent fit for the current BBSRC Responsive Mode Priority: Technology Development for the Biosciences.

This research will have significant academic impact as it will ultimately benefit not only ubiquitin researchers but those studying human biology, plants and microorganisms. This is because protein ubiquitination is such a prevalent regulatory process the potential to impinge on researchers working on seemingly disparate fields is high. We anticipate that the ability of the technology to identify aberrant E3 activity and undiscovered E3 ligases will lead to the identification of novel therapeutic targets, new biological insights and new biomarkers. Due to the emerging prevalence of protein ubiquitination in plants, analogous experiments could therefore be carried out and the outcome could lead to the identification of novel targets for disease prevention and pest resistance. Bacterial and viral pathogens also encode components of the ubiquitin system. For example, Salmonella and pathogenic E.coli encode E3-like proteins. These proteins serve as secreted effectors by subversion of the hosts ubiquitin system. In a climate where antibiotic resistance is becoming a topic of pressing concern, such proteins could be urgently needed next-generation therapeutic targets. However, there are challenges in identifying these proteins due to their low sequence similarity with their human counterparts. The unbiased nature of the proposed technology for identify E3 activity in extracts from microorganisms would enable a comprehensive inventory of E3-like proteins in a given microorganism to be determined. Indeed we have already shown that our probes detect the activity of a bacterial E3-like effector.
We will share our research findings by continuing to publish in high impact journals (i.e. Nature Chemical Biology, Nature Biotechnology, JACS). We will also present our findings at international Ubiquitin (Keystone, FASEB, EMBO) and Chemical Biology conferences (ICBS, EMBO).
To ensure broad accessibility to this technology we will distribute the developed probes (within reason) to researchers upon request. To reduce burden and costs on our part we will ensure enhanced distribution of the small molecule custom building blocks that will not be readily accessible to researchers without synthetic chemistry expertise. This will allow those with conventional protein chemistry expertise to prepare the required tools themselves. We will ensure parallel publications are available that describe the protocols in exhaustive detail allowing replication of our technology.
A patent has been filed on the technology this proposal will be built upon. This will place the University of Dundee in a position to advertise the technology to interested companies for license. The University of Dundee has demonstrated its capacity to provide services to the wider research community (e.g. kinase profiling, DNA sequencing). This will place us in a position to use our technology as a service for interested parties. The technology could also be licensed to companies. The Universities Research and Innnovation Services department actively advertise appropriate cases such as that described within. Indeed, former technology developed by myself and Jason Chin is currently under license by a commercial party.
Our MRC-PPU unit has a long-standing collaboration with Pharmaceutical companies as part of the Division of Signal Transduction Therapy (DSTT) consortium. The findings of research will be relayed to the consortium via presentations at scheduled meetings and update reports. We will therefore be in a prime position to forge collaborations with big pharma and push our technology in a more translational direction.
This project is highly disciplinary involving aspects of cell biology, chemistry and omic methodologies. The future of life science research lies in innovative combinations of these technologies and the proposed projects should provide excellent training for the 2 PDRAs that will be funded by this award.