Ubiquitylation within and beyond the DNA damage response

As we age our cells become increasingly sensitive to accumulating damage in their genetic makeup, which is formed by a complex molecule called DNA. DNA damage happens frequently, for instance through radiation, sunlight, chemicals in tobacco smoke, and even from the oxygen in the air we breathe. A rise in DNA damage is linked to some of the most severe and common problems that prevent us from healthy ageing. This includes cancer and diseases of the brain, such as loss of mental abilities. In fact, certain permanent DNA changes that affect the ability of cells to repair DNA damage, can lead to an early onset of these diseases and to premature ageing itself. With the global population ageing, the burden of age-related disorders will steadily increase. It is therefore of urgent importance that we understand better how cells prevent DNA damage. This could lead to ways to prevent these disorders and thus, contribute to healthy ageing across the lifespan.

To prevent permanent DNA damage, cells have found many different ways to repair the damaged DNA. Hundreds of proteins -the workhorses of the cell- need to quickly change their behaviour in highly organised ways, an amazing feat that is far from being properly understood. While cells can make use of an elaborate toolkit for this, the following way is especially fascinating: a little protein, known as ubiquitin, is attached to other proteins. Ubiquitin can be attached in different ways, as a single ubiquitin or as poly-ubiquitin chains made up of many ubiquitins glued together in different ways. The process of ubiquitin attachment is called ubiquitylation. The ubiquitylated proteins are recognised by other proteins, which trigger the final change in the behaviour of the ubiquitylated protein. Ubiquitylation relies on several enzyme groups, including ones known as E2s. I have recently found that several E2s play fundamental roles in repairing DNA damage. Through using novel techniques that I have been key in developing, one of the major aims of the proposal is to better understand how precisely different E2s accomplish DNA repair. This could highlight their potential as drug targets, meaning that they could be changed by a medicine to give a desirable effect. Such an effect could for instance be towards treating age-related diseases such as cancer. Moreover, my recent findings suggest that many more proteins recognise and translate ubiquitylated proteins than is currently expected. Another key aim of the proposal is therefore to analyse these ubiquitin binding proteins. By doing so the proposed work will transform our understanding of how different ubiquitylations change the behaviour of proteins in defined organised ways. These discoveries could be attractive to the commercial sector, as they have potential to be developed into general ubiquitin research tools. Since ubiquitylation is involved in almost every aspect of cell biology, the proposed work is likely to have a wide impact regarding this.

Taken together, the proposed research will increase our fundamental knowledge of how ubiquitylation regulates the DNA damage response and associated processes. This is crucial to maintaining DNA integrity. Given the significance of the DNA damage response for preventing various age-related disorders, the work may pave the way for the development of new medicines contributing to healthy ageing throughout life.

Accumulation of DNA damage and a decrease in a functioning ubiquitin system are linked to disorders associated with unhealthy ageing, such as cancer and neurodegenerative diseases. Ubiquitylation has recently emerged as a key pathway in the DNA damage response (DDR) and is executed by two E1 and ~40 E2 enzymes that team up with different sets of >600 E3 ligases to generate the ubiquitin code. Ubiquitin can be attached singly or as poly-chains with different topologies and can also be phosphorylated. Despite the importance of ubiquitin and the DDR for healthy ageing, fundamental questions remain unanswered. Thus, it is unclear how E2s create the ubiquitin code of the DDR and associated processes and how this code is translated into cellular functions by ubiquitin-binding proteins.

By integrating innovative multi-disciplinary methods and systems approaches, the proposal will start narrowing this knowledge gap: Specifically, the studies will:
(1) Define the roles of selected E2s in the DDR by delineating their associated E3(s), substrates and ubiquitylation sites. This will be enabled through a novel chemical biology technique.
(2) Identify and characterise how ubiquitin chains are decoded by ubiquitin binding proteins within and beyond the DDR, integrating a cutting-edge systems approach. This will identify novel ubiquitin binding domains, which could be used as general research tools to identify functions of underexplored ubiquitin chains.
(3) Functionally delineate novel down-stream ubiquitin binding proteins of phosphorylated ubiquitin, identified from a proteome-wide screen.

The proposal will lead to mechanistic insights into how ubiquitylation pathways regulate the DDR and associated processes. Moreover, the findings will transform our understanding of how ubiquitylations are translated into cellular events. In the long term the work may be key in defining new strategies to treat age-related diseases associated with ubiquitin and DDR pathways.

Potential beneficiaries of the proposal include the following:

Academia (main beneficiary): (1) As the work is primarily fundamental in nature, it will provide enhanced knowledge of DDR-regulated ubiquitylation pathways to scientists and clinicians interested in the biology of genome maintenance mechanisms and associated processes in human health and disease. (2) The study integrates innovative cross-disciplinary approaches and technologies that can widely be applied, thereby broadening its potential impact to any cell biologist.

Private sector: (1) The PDRA will be trained in a variety of specialist techniques, some of which are commonly used in pharma/biotech companies, which could provide the commercial sector with highly trained staff. (2) My staff will acquire various transferable skills, such as critical thinking and analytical, organisational and management skills, which could be applied in all employment sectors. (3) Inhibition of DDR enzymes can be used to treat cancer, as demonstrated by the recent approval of Olaparib, a PARP inhibitor, to treat BRCA-deficient ovarian cancers. Due to their deep yet accessible catalytic clefts, UBE2Rs represent druggable enzymes with unexplored therapeutic potential. Therefore, the results could become attractive to the biomedical industry, which should find it feasible to successfully develop suitable inhibitors against them. This could lead to anti-cancer medicines in the longer term. (4) The proposal aims to elucidate protein interaction surfaces in DDR and associated factors that allow them to bind to different ubiquitin (Ub) 'flavours'. This could highlight the potential of such surfaces as targets for small molecules. Thus, the work could become attractive to the biomedical industry, since inhibition of DDR pathways can be exploited therapeutically (see above). (5) Our work is likely to lead to new Ub binding domains specific for atypical Ub chains. Such domains are attractive as research tools to study new functions of atypical Ub chains. Thus, the findings have potential to be commercially exploited by biotech companies selling such research tools, some of which are based in the UK.

Society: (1) My group will engage with the general public through various routes (see Pathways to Impact) in order to promote the importance of science, thereby achieving societal impacts. (2) By better understanding the roles of E2 enzymes in the DDR and potentially identifying druggable protein interaction surfaces relevant to cancer and/or neurodegenerative disorders, our findings might underpin the development of new therapeutics to treat these and associated diseases. This could significantly enhance the quality-of-life for a large proportion of the population -particularly the aged- in line with BBSRC strategic priorities.

Timescales: Academic impacts will be realised throughout the program, especially in association with arising publications. To ensure swift progression towards industrial impacts, I will seek to interact regularly with industry members throughout the program, for instance at conferences. Moreover, I will hold a workshop aimed at engaging with academic and industrial end-users at an early stage. The latter will primarily be based in the UK, putting UK companies at an advantage to exploit arising commercial potential ahead of international companies. Societal impacts through public engagement will be ensured throughout the work by partaking, for instance, in regular public events organised by the University of Manchester. Potential advances towards longer-term impacts on the quality-of-life of the public depend on engagement with the commercial sector, which will be initiated as quickly as possible, as outlined above.

Collectively, it is clear that the proposal will have wide-ranging impacts on academia. Moreover, it has potential to impact on the private sector and the general public. Key steps to maximise these impacts are outlined in 'Pathways to Impact'.