Regulation of Linear Ubiquitin Signaling in Innate Immunity

Autoimmune diseases are a major burden to our society which affects a vast number of the UK population. There are about 3 million people diagnosed with conditions such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, or type I diabetes. These are long-term illnesses which persist through-out the patient's life which incur combined costs of 13 billion pound per year to our health system. A major contributing factor for the aetiology of autoimmune disease is the dysregulation of cellular immune signals, which result in an uncontrolled inflammatory response to certain stimuli. A central role in this process plays a molecule known as NFkappaB, which activates various genes required to trigger an inflammatory reaction. The function of NFkappaB is usually tightly regulated and only activated if an inflammatory response is beneficial. Since imbalanced activity of the NFkappaB pathway is a widely recognized cause for pathological inflammation, a major aim in the research area of autoimmune diseases is to understand the regulation of NFkappaB in detail. One of the main regulatory mechanisms is facilitated by an enzyme called LUBAC. This enzyme attaches a molecule chain know as linear poly-ubiquitin onto several components of the NFkappaB activation pathway. Although linear poly-ubiquitin is a potent activator for the inflammatory response, the mechanism which control the production of linear-poly ubiquitin is not well understood. This project focusses on the molecular characterisation of a recently identified molecule N4BP1 which regulates this activity. We aim to identify a detailed function of this protein and will deliver a molecular analysis how N4BP1 integrates into the process of linear poly-ubiquitin synthesis and attachment to target proteins. The findings of this research project will provide an atomic description about the regulatory role of N4BP1 which informs research and development programs aiming on the discovery of new therapeutic avenues for the intervention of autoinflammatory disease.

The inflammatory genetic programme induced by TNF mediated NFkappaB activation is tightly controlled by ubiquitylation of various components of the TNF receptor signalling complex including NFkappaB essential modifier NEMO. For example, binding and modification of NEMO with linear poly-ubiquitin chains results in subsequent migration of the transcription factor into the nucleus to trigger an inflammatory response. However, prolonged activation the TNF signalling pathway results in assembly of a proapoptotic complex which initiates cell death. The transition between pro-inflammatory gene expression and apoptosis by TNF signalling depends largely on the balanced activity of the linear ubiquitin chain assembly complex LUBAC. The importance of linear ubiquitylation in the process of NFkappaB mediated immune signaling is best demonstrated by the recent discovery of several genetic diseases which exhibit impaired LUBAC functionality that result in severe pathological consequences such as invasive pyogenic bacterial infection but also autoinflammation. Despite the prevalent role of the linear ubiquitylation machinery in innate immunity the precise factors which control and regulate signaling by LUBAC are largely unknow. We have identified the protein N4BP1 as a novel linear ubiquitin binding protein which acts as an inhibitor of LUBAC. We therefore hypothesize that N4BP1 is a negative regulator for linear ubiquitylation, a role which is in line with the recently discovered function of N4BP1 as a suppressor for cytokine production. Using a combination of structural biochemical techniques, we will elucidate the linear ubiquitin binding properties of N4BP1, determine the functional mechanism of N4BP1 mediated inhibition of LUBAC and explore the functional role of the interaction of N4BP1 and NEMO in molecular detail. The outcome of this research will provide a knowledge base for R&D programs which aim to develop new therapeutic concepts in inflammation and immunity.