Defining the roles and functional interdependencies of linear ubiquitin-related deubiquitinases and LUBAC in TNFR1 signalling

The human body is made up of billions of cells which communicate with each other. One way for cells to communicate is via messaging factors that are produced by one cell, travel through the space between cells and finally bind to the surface of another cell. The cell that receives the messaging factor will respond to the factor as appropriate. Such factors are particularly important in the communication of different types of immune cells with each other but also in the way immune cells and other cells communicate with each other. Usually these factors stimulate a response from the receiving cell which alerts the immune system. In some cases the receiving cell is killed by the communication factor. However, the dying cell itself responds by sending out powerful signals in the form of specific factors that are released from the dying cell. These highly potent communication factors are known as "damage" or "danger" signals and represent some of the most powerful means by which the immune system can be engaged.

One of the most important factors in cell-to-cell communication is the tumour necrosis factor (TNF). TNF is crucial for fighting off one of the deadliest infections known to humankind which is caused by infectious agent that causes tuberculosis. However, given the extraordinary power of TNF, it is perhaps not surprising that this factor can also cause severe damage when it is deregulated. This is exemplified by the discovery that TNF is causative for several autoimmune diseases including rheumatoid arthritis, Crohn's disease, and psoriasis. Consequently, patients suffering from these diseases often benefit from treatment with drugs that inhibit TNF. These drugs are very costly and they also block the effects of TNF completely rather than only the disease-causing consequences of TNF without interfering with the desirable activities of TNF. The aim of our research programme is to better understand the biology of TNF and how stimulation of a receiving cell by TNF leads to different outcomes so that, in the future, we can target the disease-causing aspects of the biology of TNF more precisely.

Depending on the receiving cell's make-up, TNF can either trigger a signal that alerts other cells, including immune cells, but keeps them intact or it can trigger cell death which can then alert the immune system in a different way by the release of the afore-mentioned danger signals. The precise outcome of TNF binding to the receiving cell is determined by factors within the receiving cell and we recently identified three factors which regulate this outcome. These factors have a common function and were previously not known to act together to define the outcome of a cell's stimulation by TNF. Our recently performed experiments led us to conclude that each one of these factors, apart from serving a common function in the regulation of the outcome of TNF stimulation, may also have "private" functions in the so called "TNF signalling". The aim of the research proposed here is to identify which of these functions are served by these factors in common and how do they fulfil their function specifically but also which functions are served by them "privately". We also want to uncover which parts of these proteins are responsible for the "common" and "private" functions, respectively, and how disrupting defined parts of these factors affects the cell's response to stimulation by TNF. We want to understand this at the molecular and mechanistic level but also in cellular systems and, ultimately, in the living organism. We envisage that a better understanding of the functional outcomes of stimulation by TNF, which is decisively controlled by the factors we propose to study here, will contribute to being able to distinguish the desired from the undesired functions of TNF molecularly and, consequently, to the development of therapeutic strategies which allow us to specifically target those TNF functions which cause disease.

Ubiquitination events are major regulators of immune receptor signalling. Tumour necrosis factor (TNF) is a crucial cytokine for the development of an effective immune response to infections. It is therefore important that TNF can act in the physiologically desired manner. However, in many diseases, including several auto-immune diseases but also in certain inflammatory-driven cancers, TNF signalling is perturbed. The main receptor for TNF is TNF receptor 1 (TNFR1). TNFR1 signalling is exquisitely regulated by ubiquitination and de-ubiquitination events. We and others have recently shown that LUBAC, the only known ubiquitin ligase specific for the generation of M1-linked ubiquitin chains (linear ubiquitin) is recruited to the TNFR1 signalling complex (TNFR1-SC) where it is required for optimal gene-activatory signalling whilst limiting TNF-induced cell death. Maintaining the balance between these two outcomes is crucial for both, TNF's role in immunity to infection and in preventing it from inducing auto-immunity. In this proposal, we aim to understand the role of LUBAC and LUBAC- or linear-ubiquitin-related deubiquitinases (DUBs) in the regulation of the different TNF signalling outputs, i.e. gene activation and cell death. In particular, we will study how LUBAC and linear ubiquitination regulate these three DUBs and how these DUBs regulate and affect linear ubiquitination in TNFR1 signalling and the various functional outputs of TNFR1 stimulation. We are also interested in studying the inter-dependencies of these DUBs in regulating LUBAC and linear ubiquitin. Defining the specific and inter-dependent roles of these factors shall reveal important aspects of the biology of TNF and will hopefully inform future therapeutic strategies which we hope will rely on specific targets that only interfere with certain, pathological aspects of TNFR1 signalling instead of systemically inhibiting TNF which is the current standard approach in the clinic.

Our proposal is aimed at providing a better understanding of the interplay between the three most prominent de-ubiquitinases involved in TNF signalling (CYLD, OTULIN and A20), LUBAC and linear ubiquitin. This will likely result in the identification of previously unrecognised molecular and functional aspects of inflammatory pathologies. This contribution will be vital for the progression of the research fields of cell death, inflammation and immunity. The expected new insight generated through this research will be beneficial for the research community but may also become relevant for the commercial sector as it could lead to the development of new treatment strategies.

The academic field thrives on the discovery of novel aspects of key biological processes. Therefore an immediate beneficiary of our work is academia. Our research will further contribute to filling the gaps in our current understanding of ubiquitin-regulated and -independent pathological inflammation. This will also help the postdoctoral scientists working on this project promote their independent careers. Furthermore, in understanding the individual and cooperative functions of CYLD, OTULIN and A20 with respect to LUBAC and linear ubiquitin in TNFR1 signalling we will potentially be able to target underlying signalling pathways more specifically and adequately which will likely lead to future projects which we envisage should also include new projects on targeted drug development.

Therefore, in the long term, the acquired knowledge and strategies will likely help us devise novel strategies for the treatment of inflammatory diseases that involve TNF signalling and possibly other immune signalling pathways. Hence, the commercial sector involved in drug development, i.e. the biotechnology and pharmaceutical sectors, will likely benefit from the new insight provided by the research proposed here which, ultimately, is aimed at translating into improved treatments for patients suffering from inflammation-related diseases. Additionally, it is likely that important insight may be gained with respect to HOIP-, HOIL-1-, CYLD-, A20, and OTULIN-mutation-driven diseases (e.g. Brooke-Spiegler syndrome and OTULIN-related autoinflammatory syndrome - ORAS) could be drawn from our study which will hopefully help these patients in the longer term.