Investigating the targets and biological roles of the deubiquitylase USP43

The cells in our body must be able to sense and respond to changes in their environment (hormones, UV damage, toxins) to maintain the healthy function of tissues and organs. Failure to do so progressively undermines cellular 'fitness' contributing to age-related declines in cell and tissue function that drive the normal ageing process and can contribute to age-related diseases such as chronic inflammation, cancer and dementia.
Cells respond to tissue damage or infection by increasing the abundance of key proteins and enzymes that mitigate that damage. They do this through the activation of intracellular signalling pathways which transmit information into the cell. When a cell receives this signal the genes that code for enzymes are 'read' by 'transcription factors', discrete proteins that bind to DNA and transcribe the DNA information into RNA molecules, which are in turn 'translated' into the relevant proteins and enzymes. One such transcription factor, called 'NFkappaB' (NFkB), coordinates cellular responses to an inflammatory signal called 'TNF'. When cells are exposed to TNF, NFkB becomes activated as a result of phosphorylation - the attachment of phosphate groups; this requires two enzymes called the IkB kinase or IKKs. We have deleted the genes for both IKKs in human colon cells using CRISPR gene editing. By analyzing the changes in abundance of RNA molecules we found that TNF-NFkB signalling activates the gene that codes for a protein called USP43. TNF cannot increase USP43 abundance in cells that lack IKKs.
We also noted that cells that lack IKK have increased cell-to-cell contacts (i.e. closer physical contact with neighbouring cells) and increased abundance of a protein called E-cadherin that mediates cell:cell adhesion signals. We therefore examined how cells respond to increasing cell density, which promotes cell:cell contacts. This led us to discover that USP43 abundance also increases with increasing cell density and that USP43 is found in cells close to E-cadherin at sites of cell-cell contacts.
Formation of correct cell:cell contacts is vital for the formation and maintenance of complex tissues. If it fails it can cause inflammation, including inflammatory bowel disease (IBD). Indeed, genetic defects in components of the TNF-IKK-NFkB pathway and E-cadherin have both been shown to contribute to IBD syndromes so it is very striking that USP43 abundance is controlled by both pathways and is found at sites of cell:cell contact in colon cells. Indeed, recent data from another lab has found mutations in the gene for USP43 which impair USP43 activity in patients with inflammation.
For these reasons we are interested in the function of the USP43 protein, which belongs to a family of enzymes that cleave a protein called ubiquitin from other proteins. Many proteins in our cells are 'tagged' by the addition of ubiquitin and this changes the properties of such proteins, regulating their activity, directing them for destruction or directing them to specific compartments within the cell. USP43 reverses the addition of ubiquitin to proteins. We suspect that USP43 controls the activity or abundance of proteins that are critical for TNF-IKK-NFkB inflammatory signalling and E-cadherin signalling.
In this project we will define how USP43 abundance is controlled, identify the targets of USP43 (those proteins that USP43 removes ubiquitin from) and other USP43 interacting proteins that may control its functions. We will delete the USP43 gene from human cells so that we can assess the role of the USP43 protein in regulating inflammatory signalling, cell:cell contacts, cell survival and cell division. The results will shed new light on how inflammation and tissue structure are controlled and may tell us whether USP43 is a possible new drug target for inflammatory disease.

Deubiquitylases (DUBs) reverse protein ubiquitylation, controlling protein fate to influence cell physiology. DUBs have emerged as attractive drug targets as they are proteases with well-defined catalytic sites and some are de-regulated in specific diseases. However, many DUBs, including USP43, are poorly characterised in terms of their subcellular location, regulation and targets as well as the biological processes they control. USP43 has been implicated in the control of NFkB signalling and, independently, has been suggested to promote epithelial-to-mesenchymal transition (EMT); in both cases the underlying mechanisms are unknown.

We have now found that:
1. USP43 is expressed in response to TNFa in an IKK-dependent manner; thus it is a novel NFkB target gene.
2. USP43 is also expressed with increasing cell density suggesting it may be regulated by the Hippo-YAP pathway. Indeed, YAP seems to repress USP43.
3. USP43 colocalises with E-cadherin at the plasma membrane in human colon epithelial cells. E-cadherin is a cell-cell adhesion molecule that coordinates cell epithelial differentiation, survival, proliferation and supports the epithelial barrier.

TNF-NFkB signalling and E-cadherin are both deregulated in gastrointestinal inflammation, including inflammatory bowel diseases, where TNFa drives loss of E-cadherin and EMT to compromise the epithelial barrier. Our new data suggest that USP43 is positioned to regulate this pathway.
We will investigate: (i) how USP43 expression is regulated by TNF-NFkB signalling and epithelial cell-cell contacts; (ii) identify the targets of USP43 using BioID-based proximity labelling and mass spectrometry; (iii) define the role of USP43 in regulating TNF-NFkB signalling and (iv) define the role USP43 in cell density-dependent growth control, EMT and epithelial barrier function.
This is a fundamental biology study; however it may have far reaching implications for colleagues studying inflammatory disease and cancer.

The primary impact will come from new knowledge of mechanisms of signal transduction, related to the role of USP43 (see Academic beneficiaries). Impacts on other stakeholders:-

1. BBSRC: meeting national strategic research priorities.
The regulation of USP43 by cell-cell contact signalling is relevant to developmental decisions that define tissue integrity. The identification of UPS43 as a novel target of NFkB is relevant to normal and pathological inflammation, including IBD, the incidence of which is increasing in the ageing population. Thus within the BBSRC Strategic Plan this research lies at the heart of Strategic Research Priority 3 - Bioscience For Health - and is relevant to the Societal Grand Challenge of 'maintaining health across the whole lifecourse' and the Key Priority 'Generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse'. In addition, this work 'will provide new insights to potential strategies for health monitoring and intervention, including drug targets and pharmaceuticals' consistent with the aspiration that 'basic bioscience funded by BBSRC underpins the pharmaceutical and healthcare industries'.

2. Industry - by enhancing the research capacity and knowledge of businesses and organisations.
The identification of USP43 as a DUB that coordinates NFkB and cell density dependent signalling will be of interest to many Biotech/Pharma companies working in inflammation, IBD and cancer. Many companies are interested in DUBs as drug targets and we are already collaborating with MISSION Therapeutics, based on the Babraham Research Campus so we already have a potential 'pathway to impact'. However, other BioPharma companies are also operating in this space (e.g., FORMA, Cancer Research Technology). We will work with the Babraham Institute Commercialisation Manager and our wholly-owned trading arm BI Enterprise to manage any resultant IP and all interactions with industry.

3. Healthcare sector, 3rd sector charities and patients: translation of research into the clinic.
USP43 is a druggable target that is implicated in inflammatory signalling which contributes to infirmity in old age. In addition, USP43 expression increases with cell density and USP43 colocalises with E-cadherin at epithelial cell:cell contacts suggesting that it may regulate EMT. These processes are deregulated in a variety of clinical conditions that involve cell migration and invasion including placentation defects, preeclampsia and cancer metastasis. Thus, the basic biology of USP43 may prove to be of interest to a variety of disease charities, healthcare professionals and patients. We will liaise with these groups through local research networks.

4. Training: generating a skilled workforce.
This project will provide further training for key researchers (PDRA & Cook) in new scientific skills in growth areas (proteomics, genomics, bioinformatics). The PDRA will learn about drug discovery research, providing training for their future contribution to UK science & economic output in either the academic or commercial sector. They will receive training in key transferable skills such as communication through conference attendance and involvement in PE and KEC activities.

5. Science & Society: influencing and informing policy and increasing public understanding of science.
New knowledge in this area may impact patients (e.g. IBD patients, other inflammatory diseases etc.) so we will share our knowledge and results with them. Public health is a key policy area, so policy-makers may find our research of interest. Inspiring the next generation of researchers, through our passion and knowledge of curriculum-relevant science: students at KS3 learn about cells as a fundamental unit of living organisms; at KS4 students explore the relationships between health and disease. Also, all students have to learn how to work scientifically, developing critical thinking skills.