The P2X7 Interactome: Protein interactions in the P2X7 C-terminus and their role in inflammatory signalling
Lead Research Organisation:
University of Bath
Department Name: Pharmacy and Pharmacology
Abstract
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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 rheumatoid arthritis, which currently affects around 400000 people in the UK alone. Research into inflammation and its causes is therefore 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 close a significant gap in our knowledge, and 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 therapeutic benefit in the future.
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 made a major advance in 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 and understandable by 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 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, 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 rheumatoid arthritis, which currently affects around 400000 people in the UK alone. Research into inflammation and its causes is therefore 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 close a significant gap in our knowledge, and 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 therapeutic benefit in the future.
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 made a major advance in 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 and understandable by 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 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, particularly in the ageing population, reducing the burden of age-related disease on health services worldwide.
Organisations
People |
ORCID iD |
Amanda MacKenzie (Principal Investigator) |
Publications
Stokes L
(2016)
Editorial: Roles of Ion Channels in Immune Cells.
in Frontiers in immunology
Description | Inflammation is an important physiological response to infections and injury. The body responds by detecting the release of danger signals such as extracellular ATP sensed by cell surface P2X7 receptors. It is well established that P2X7 receptors communicate inflammatory signals in cells of the immune system. The P2X7 receptor is formed from three protein subunits with portions that reside inside the cell. This project aimed to understand how P2X7 receptors interact with other proteins inside the cell ('the P2X7 interactome') and how this interaction regulates inflammation. The primary aim of the grant was to understand the initial steps in the inflammatory signalling cascade by isolating the internal portion of the P2X7 receptor (240 amino-acid C-terminal domain (CTD), identify interacting proteins from isolated cell extracts and understand how these proteins regulate inflammatory responses. A secondary aim was to study the structure and organisation of the P2X7 CTD. As part of this project 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 have used mouse macrophages and a model cell line (HEK293) expressing an engineered P2X7 protein to confirm interacting proteins identified binding to the CTD. Initial studies aimed to measure interaction using proteins extracted from cells but failed to consistently detect proteins binding onto the P2X7 protein. We progressed to using a 'fluorescent proximity ligation assay' to detect the interaction between two proteins in intact cells. We have confirmed known interactors (pannexin 1), studied the regulation of interaction and potential regulation of inflammatory signalling pathways. Some of these results have been presented as a selected oral presentation at the Purines Keystone Symposia, Vancouver 2016. |
Exploitation Route | These findings confirm the existence of a P2X7 receptor interactome in intact cells. This will be important for our understanding of the properties of P2X7 receptors in inflammatory cells and other cell types. New P2X7 receptor constructs developed in the project will be useful for continuing studies of the structure, function and interactions of the P2X7 receptor protein. The P2X7 Interactome database is a very useful repository of information about protein interactions and P2X7 function, verbal feedback from other P2X receptor researchers indicate that it is useful. The P2X7 interactome website has been used educationally for undergraduate and postgraduate students at the University Bath. It is planned that the P2X7 interactome website will continue to be maintained and developed at the University of Cardiff. |
Sectors | Education,Pharmaceuticals and Medical Biotechnology |
URL | http://www.keystonesymposia.org/16J5 |
Description | Our findings have not been used to date in a quantifiable way |
Title | P2X7 interactome website |
Description | Valuable database for the P2X7 research community - quick links to published work. Valuable learning resource for students studying the area |
Type Of Material | Database/Collection of data |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | Cited in research publications and reviews https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693442/ https://www.frontiersin.org/articles/10.3389/fchem.2018.00248/full |
URL | http://p2x7.co.uk |
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 | P2X7 Interactome Website |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | The P2X7 Interactome Website was launched in Sept 2013. Announcements have been made via the UK Purine Club and associated groups on Linkedin. The P2X7 Interactome Website collates all the published reported interactors with P2X7 (links to paper, sequence, structural information). Information regarding the properties of P2X7 receptors (structure, splice variants, downstream signaling pathways, pharmacological properties) is provided. An additional section for uploading methods is included. Open Access website no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |
Description | Poster presentation at Purines 2014, Bonn, Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research and patient groups |
Results and Impact | Poster presentation ('The P2X7 Interactome' by Marta Radwan in Purines 2014, Bonn, Germany from 23rd - 27th July. An International Conference on Signalling, Drugs and Targets in Nucleotides, Nucleosides and Nucleobases. This was an international conference with 231 poster presentations, 8 plenary lectures and 34 symposium sessions. Provided oral feedback on the use of the P2X7.co.uk website. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2014 |
URL | http://domains.conventus.de/fileadmin/media/2014/purines/pdf/AbstractBook.pdf |
Description | The P2X7 Interactome - poster 2013 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | poster presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Poster presented at the UK Purines Meeting at Jesus College, Cambridge on 16th December 2014. This was a National 1 day conference with international invited speakers (Adinolfi, Pelegrin, Dixit and Novak) organized by the UK Purine Club. In total 59 delegates. Poster no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |