The P2X7 Interactome

Inflammation is a normal, healthy physiological response to infection or injury. However, in countries with ageing populations, such as the UK, the unwanted and painful consequences of age-related chronic inflammation, in conditions such as arthritis, cardiovascular disease and Alzheimer's disease, have a significant negative effect on health and quality of life. Arthritis, for example, currently affects greater than 2 million people in the UK alone. This is why research towards a molecular understanding of inflammation and inflammatory signalling is of great impact to human health worldwide, and fits within the BBSRC strategic priority of ageing research: lifelong health and wellbeing. The purpose of this research is to understand precisely how immune cells such as macrophages respond to inflammatory signals, by discovering and characterising a key step in the signalling pathway. Pro-inflammatory signals are recognised by receptors on the cell surface of macrophages. Some pro-inflammatory signals are key cellular metabolites such as ATP, because when they are released from cells, it indicates that cells are dying due to stress, infection or tissue injury. Released ATP is recognised by P2X7 receptors, and this leads to a downstream signalling cascade, that amplifies the pro-inflammatory signal and can lead to a full-scale inflammatory response (characterised by swelling and pain in the affected area). The P2X7 receptor contains 3 large domains; one which recognises ATP (on the outside of the cell), one which allows ions to enter the cell upon activation (embedded in the cell membrane), and one which switches on a signalling cascade upon activation (inside the cell). While we have some information about how ATP binds to the receptor, and how ions can enter the cell, we do not understand how the signalling cascade is switched on. We hypothesize that the intracellular domain of P2X7 must have a defined 3D structure and organisation that enables it to interact with other signalling proteins, but we do not know what it is, or what the identities of the interacting proteins are. To understand how the inflammatory process functions in healthy humans, and to devise strategies to treat unwanted inflammation in the ageing population, it is vital that we understand the structure, organisation and interactions of the intracellular domain of P2X7. A good way to understand the structure and functions of a protein domain is to study it in isolation from other parts of the protein. We have recently been able to make and purify a major portion of the P2X7 intracellular domain, the intracellular C-terminus, in yeast. We aim to exploit this advance, and use our purified protein in two major ways. First, we want to investigate the structure and organisation of the isolated protein, by looking at which parts are important for inflammatory signalling, and which parts are important for holding its structure together. Second, we want to use the C-terminus as bait, to fish for proteins which interact with it, in intracellular extracts from macrophages. We can then isolate the interacting proteins and identify them, discovering novel interacting protein partners for the C-terminus of P2X7. Once we have discovered how the P2X7 C-terminus structure is organised, and identified its interacting partners, we can test the effects of disrupting these interactions on pro-inflammatory signalling pathways in macrophages. This will enable us to understand precisely how P2X7 activation switches on inflammatory signalling pathways, and significantly advance our understanding of inflammatory processed in human health.