The P2X7 Interactome: Protein interactions in the P2X7 C-terminus and their role in inflammatory signalling
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Biosciences
Abstract
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.
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.
Technical Summary
ATP is released by cells under a variety of conditions, including mechanical stress, infection or tissue injury, where it activates the P2X7 receptor, initiating an inflammatory signalling cascade involving calcium influx, opening of a dye-permeable plasma membrane pore, reactive oxygen species generation, inflammasome activation, cytokine release and cell death via apoptosis or necrosis. Many of these events are regulated by the 250 amino-acid C-terminal domain, yet surprisingly little is known about the structure, organisation and role of the P2X7 C-terminal domain in macrophage signalling, and the key first step in pro-inflammatory signalling following P2X7 activation remains to be elucidated.
Recently, we have been able to express the C-terminal domain of rat P2X7 in yeast, purify folded protein, and demonstrate that it forms known interactions. We aim to exploit this important breakthrough to study the structure, organisation and interacting partners of the mouse P2X7 C-terminus. We will use the purified C-terminus in pull-down assays using mouse bone marrow-derived macrophage lysates, to discover novel intra- and inter-molecular interactions (the P2X7 Interactome). We will then feed this data into co-expression studies in HEK cells, to confirm interactions by immunoprecipitation and proximity ligation assay, and measure the effects of P2X7 C-terminal truncations and mutants on both ion channel function (using patch clamp electrophysiology) and signalling pathways (using dye uptake assays). We will then translate our studies into mouse macrophages, using si-RNA knockdown of interacting partners, multiplex assays for cytokine release and assays for cell death via apoptosis (Annexin V labelling, cytochrome c release) or necrosis (lactate dehydrogenase release). In this way we will not only define the structural organisation of the P2X7 C-terminal domain, but also identify the key first step in pro-inflammatory signalling following P2X7 activation.
Recently, we have been able to express the C-terminal domain of rat P2X7 in yeast, purify folded protein, and demonstrate that it forms known interactions. We aim to exploit this important breakthrough to study the structure, organisation and interacting partners of the mouse P2X7 C-terminus. We will use the purified C-terminus in pull-down assays using mouse bone marrow-derived macrophage lysates, to discover novel intra- and inter-molecular interactions (the P2X7 Interactome). We will then feed this data into co-expression studies in HEK cells, to confirm interactions by immunoprecipitation and proximity ligation assay, and measure the effects of P2X7 C-terminal truncations and mutants on both ion channel function (using patch clamp electrophysiology) and signalling pathways (using dye uptake assays). We will then translate our studies into mouse macrophages, using si-RNA knockdown of interacting partners, multiplex assays for cytokine release and assays for cell death via apoptosis (Annexin V labelling, cytochrome c release) or necrosis (lactate dehydrogenase release). In this way we will not only define the structural organisation of the P2X7 C-terminal domain, but also identify the key first step in pro-inflammatory signalling following P2X7 activation.
Planned Impact
Relevance to BBSRC strategy
Research towards a molecular understanding of inflammation and inflammatory signalling is of great impact to human health worldwide. While inflammation is a healthy response to tissue infection or injury, older people tend to be more likely to suffer from pain and a reduced quality of life with conditions of chronic inflammation such as arthritis, cardiovascular disease and Alzheimer's disease. Arthritis alone currently affects more than two million people in the UK; this is why research into inflammation and its causes is a direct fit with the BBSRC strategic priority of ageing research: lifelong health and wellbeing.
The aim of the 'P2X7 Interactome' project is to understand the molecular basis for pro-inflammatory signalling via P2X7 receptor activation. We still do not understand how activated P2X7 receptors are able to initiate a cascade of downstream events, such as the release of pro-inflammatory cytokines and cell death. Finding out which regions within the P2X7 intracellular C-terminal domain are important for signalling, and what signalling proteins they interact with, will be of major impact to both the P2X and inflammation research fields. We will discover new protein-protein interaction targets, which will enable the screening of small molecules designed to uncouple of P2X7 activation from pro-inflammatory signalling, which may be of significant therapeutic benefit.
Who will benefit from this research?
The main non-academic beneficiaries from the 'P2X7 Interactome' research include the pharmaceutical industry and the general public. The research detailed in this proposal will lead to both a greater understanding of how P2X7 activation couples to inflammation, and also the identification of potential targets for therapeutic intervention. Several selective P2X7 antagonists have already been developed and are in clinical trials for the treatment of inflammatory diseases. These molecules all target the extracellular, ligand-binding portion of the molecule, and block the function of the ion channel as well as downstream signalling. Drugs designed to target protein-protein interactions in the C-terminal domain will have the advantage that ion channel function is uncompromised, while signalling is blocked, and so may have fewer side-effects and enhanced specificity.
How will they benefit from this research?
1. Knowledge
At the end of this project, we aim to understand how P2X7 activation leads to downstream signalling. We will have transformed our understanding of inflammation, and provided new drug targets which we will hope to exploit in collaboration with the pharmaceutical industry. In addition, we will set up and maintain a dedicated 'P2X7 interactome' website, which will contain information about known P2X7 interacting proteins and their roles in health and disease, which will be fully accessible to non-scientists.
2. People
The PDRA employed on this project will receive a comprehensive training in protein expression, purification, biochemistry and cell biology and this will contribute greatly to their individual career development. During the course of the project, they will also disseminate their knowledge to both undergraduate and postgraduate students working in the laboratory. The PI has recently secured a BBSRC Quota PhD student working on a similar protein expression project, whose training will also benefit from working closely with the PDRA.
3. Improvement of health and quality of life
The results of this research will lead to greater understanding of the role of P2X7 receptors in inflammation and human health. We hope that, by providing novel targets and ideas for therapeutic intervention, the development of P2X7-selective anti-inflammatory drugs will be accelerated, which will provide significant benefits for human health and quality of life, particularly in the ageing population, reducing the burden of age-related disease on health services worldwide.
Research towards a molecular understanding of inflammation and inflammatory signalling is of great impact to human health worldwide. While inflammation is a healthy response to tissue infection or injury, older people tend to be more likely to suffer from pain and a reduced quality of life with conditions of chronic inflammation such as arthritis, cardiovascular disease and Alzheimer's disease. Arthritis alone currently affects more than two million people in the UK; this is why research into inflammation and its causes is a direct fit with the BBSRC strategic priority of ageing research: lifelong health and wellbeing.
The aim of the 'P2X7 Interactome' project is to understand the molecular basis for pro-inflammatory signalling via P2X7 receptor activation. We still do not understand how activated P2X7 receptors are able to initiate a cascade of downstream events, such as the release of pro-inflammatory cytokines and cell death. Finding out which regions within the P2X7 intracellular C-terminal domain are important for signalling, and what signalling proteins they interact with, will be of major impact to both the P2X and inflammation research fields. We will discover new protein-protein interaction targets, which will enable the screening of small molecules designed to uncouple of P2X7 activation from pro-inflammatory signalling, which may be of significant therapeutic benefit.
Who will benefit from this research?
The main non-academic beneficiaries from the 'P2X7 Interactome' research include the pharmaceutical industry and the general public. The research detailed in this proposal will lead to both a greater understanding of how P2X7 activation couples to inflammation, and also the identification of potential targets for therapeutic intervention. Several selective P2X7 antagonists have already been developed and are in clinical trials for the treatment of inflammatory diseases. These molecules all target the extracellular, ligand-binding portion of the molecule, and block the function of the ion channel as well as downstream signalling. Drugs designed to target protein-protein interactions in the C-terminal domain will have the advantage that ion channel function is uncompromised, while signalling is blocked, and so may have fewer side-effects and enhanced specificity.
How will they benefit from this research?
1. Knowledge
At the end of this project, we aim to understand how P2X7 activation leads to downstream signalling. We will have transformed our understanding of inflammation, and provided new drug targets which we will hope to exploit in collaboration with the pharmaceutical industry. In addition, we will set up and maintain a dedicated 'P2X7 interactome' website, which will contain information about known P2X7 interacting proteins and their roles in health and disease, which will be fully accessible to non-scientists.
2. People
The PDRA employed on this project will receive a comprehensive training in protein expression, purification, biochemistry and cell biology and this will contribute greatly to their individual career development. During the course of the project, they will also disseminate their knowledge to both undergraduate and postgraduate students working in the laboratory. The PI has recently secured a BBSRC Quota PhD student working on a similar protein expression project, whose training will also benefit from working closely with the PDRA.
3. Improvement of health and quality of life
The results of this research will lead to greater understanding of the role of P2X7 receptors in inflammation and human health. We hope that, by providing novel targets and ideas for therapeutic intervention, the development of P2X7-selective anti-inflammatory drugs will be accelerated, which will provide significant benefits for human health and quality of life, particularly in the ageing population, reducing the burden of age-related disease on health services worldwide.
Organisations
Publications
Fowler BJ
(2014)
Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity.
in Science (New York, N.Y.)
| Description | Inflammation is a healthy physiological response to infection and injury. Under these conditions, high concentrations of normally intracellular molecules are released by dead and dying cells at sites of infection or injury, and these molecules are sensed by protein receptors on cells of the immune system. One such molecule, ATP, is sensed by P2X7 receptors, initiating a signalling cascade that leads to a series of pro-inflammatory cellular responses. The primary aim of this grant was to understand the first steps in this inflammatory signalling cascade, by isolating and identifying the protein molecules which interact directly with the intracellular signalling domain (240 amino-acid C-terminal domain (CTD)) of the P2X7 receptor. A secondary aim was to study the structure and organisation of the P2X7 CTD. As part of our work we developed the 'P2X7 Interactome' website to list all the published P2X7-interacting proteins (>50 are known, but it is not known whether or not they bind directly to P2X7, or where they bind), which has been useful for the P2X research field. We also collaborated with the group of Jayakrishna Ambati (Kentucky, USA), finding that reverse transcriptase inhibitors, drugs normally used to treat HIV infection, were effective in treating age-related macular degeneration (a currently incurable disease) via a P2X7 signalling-dependent mechanism (work published in Science). Although we were able to detect a physical interaction between mouse P2X7 and a known binding partner, pannexin 1, we were unsuccessful in identifying novel or direct P2X7-interacting proteins, mainly due to a high degree of non-specific protein binding in our interaction assays. We did, however, develop a number of tools which will be useful in future functional, structural and interaction studies. These include full-length and CTD mouse P2X7 protein constructs bearing N-terminal Halotag purification tags, full-length rat and mouse P2X7 bearing C-terminal eGFP and mCherry detection tags, and FLAG-His-tagged mouse pannexin-1, all for expression in mammalian cells. Work is still continuing on the secondary aim of the grant, to analyse the structure and organisation of the P2X7 CTD. At present we have generated a variety of expression constructs for this domain in bacteria, yeast, insect and mammalian cells. Our preliminary data shows that an internal sub-fragment of the CTD is highly expressed as a soluble, folded protein in E. coli, and efforts to determine the structure of this domain are ongoing. In addition, we have achieved high levels of expression of the full-length CTD coupled to eGFP in insect cells. Finally, with the aid of a new PhD studentship (Mr Ahmad Jaradat), we are investigating the role if the CTD in P2X7 signalling using a combination of mutagenesis and functional assay, to look at which regions are responsible for the various signalling functions. |
| Exploitation Route | The P2X7 Interactome database is a very useful repository of information about protein interactions and P2X7 function, and is already well-used by Undergraduate and Postgraduate students in Cardiff University as a starting point to find links to published research. We hope to maintain and develop this resource in future; verbal feedback from other P2X receptor researchers indicates that it is perceived as useful to the research field as a whole. Work towards developing reverse transcriptase inhibitors as therapies for macular degeneration continues in the lab of Jayakrishna Ambati; this would represent the first effective treatment for the dry form of the disease. The P2X7 constructs that we have generated will be useful in continuing studies of the structure, function and interactions of the receptor. |
| Sectors | Education,Pharmaceuticals and Medical Biotechnology |
| Description | Our findings have not been used to date in a quantifiable way |
| Description | PhD Scholarship To Mr Ahmad Jaradat |
| Amount | £85,500 (GBP) |
| Organisation | Mutah University, Jordan |
| Sector | Academic/University |
| Country | Jordan |
| Start | 01/2017 |
| End | 12/2020 |
| Title | P2X7 Interactome website |
| Description | A public database of all published P2X7-interacting proteins |
| Type Of Material | Database/Collection of data |
| Year Produced | 2013 |
| Provided To Others? | Yes |
| Impact | Valuable database for the P2X7 research community - quick links to published work. Valuable learning resource for students studying the area |
| URL | http://www.p2x7.co.uk |
| Description | Biology Rocks! |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | Yes |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Exhibitions were set up at the National Muesum of Wales and parents and children attended the exhibitions. None |
| Year(s) Of Engagement Activity | 2014,2015 |
| Description | Invited Seminar at RNHRD Hospital, Bath, UK; 2nd Dec 2014 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Participants in your research and patient groups |
| Results and Impact | Talk entitled 'Danger Signals, Ion channels and Inflammasomes' that disseminated information regarding the BBSRC P2X7 interactome project and the recently funded ALERT14 equipment grant. Sparked discussion with clinicians regarding the role and regulation of inflammasomes in clinical conditions. No impacts yet to date but may influence future clinical collaborations. |
| Year(s) Of Engagement Activity | 2014 |
| Description | Keystone symposia Invited oral presentation |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Selected oral presentation at Keystone Symposia Purinergic signaling joint with Cancer Immunotherapy: Immunity and Immunosuppression Meet Targeted Therapies in Vancouver Canada. Presentation to international audience in the purinergic signaling field and lead to discussion around proximity ligation assays. |
| Year(s) Of Engagement Activity | 2016 |
| URL | http://www.keystonesymposia.org/16J5 |
| Description | Learn About Life - Primary School visits to the University |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | Yes |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Primary school children from schools in the region (30 per class, 6 Schools) attended the University for demonstrations of ongoing research. Our demonstration was about the power of yeast, and the students got the opportunity to do an experiment, which sparked questions and discussions afterwards. School children saw an experiment in action and were excited to see the results. |
| Year(s) Of Engagement Activity | 2014 |