MICA: Is PI3Kgamma signalling organised in distinct membrane nano-domains?

Lead Research Organisation: Babraham Institute
Department Name: Signalling

Abstract

Neutrophils are the major white blood cell and are involved in fighting bacterial and fungal infections. Patients who have long term deficiencies in neutrophil function suffer from life-threatening chronic infections and usually die early. Neutrophils perform this role through their ability to detect potential pathogens or zones of inflammation, migrate towards them and ingest (phagocytose) and kill the pathogens. Pathogen killing is performed by a battery of anti-microbial agents including highly reactive chemical species and enzymes that are released into the vicinity or into the small digestive vacuole formed inside the neutrophil once microbes have been ingested. It is increasingly accepted that neutrophils can also be key culprits in a number of inflammatory diseases, such as rheumatoid arthritis. In these situations excessive neutrophil accumulation at the site of inflammation only makes things worse as the intense production of anti-microbial agents causes collateral damage to neighbouring cells and inflammation. This results in further neutrophil recruitment, leading to a long-term problem. The intracellular processes that control these neutrophil behaviours have been studied for many years because of their central role in immunity and in disease. In principle, some of the intracellular processes may be more important in the disease processes than they are for the immune response and hence drugs targeting them may act as anti-inflammatories whilst sparing immune function. Such compounds are much needed as current anti-inflmmatories are limited and have side-effects.
Our project addresses how these intracellular processes in neutrophil function in both health and disease. We focus on a specifc key player in these molecular events called PI3K-gamma (phosphoinositide 3-kinase) that we first discovered in the early 1990s. It is now accepted to be a potential target for novel anti-inflammatories with many major companies screening/developing new inhibitors. PI3Kgamma is found as a complex of 2 proteins; p110gamma and either a p101 or p84 regulatory subunit. All of the drugs so far developed against PI3K-gamma have been designed to inhibit the p110-gamma protein because it is always present. The relative roles of the 2 regulatory subunits are not completely understood: how they are different in terms of properties and functions and whether it might be case that one is more or less involved in pro-inflammatory v immune responses and may hence may be a better target for the development of drugs in the long term. Our recent work (unpublished) shows that in mice p84 and p101 have unique important roles in neutrophils and in this grant we hope to refine this idea and understand how they work differently and whether they offer better opportunities as drug targets.

Technical Summary

PI3Kgamma is a member of a small family of signalling enzymes, structurally distinct to the larger family of protein kinases, which operate in many receptor-sensitive networks. It has a very tissue specific distribution and is abundant in neutrophil-like cells. PI3Kg KO mice are normal under animal-house conditions but PI3Kg is required for a range of inflammatory reactions in vivo. These factors have driven large investment in the development of PI3Kg inhibitors as novel anti-inflammatories. PI3Kg is a heterodimer of a p110g catalytic subunit and either a p101 or p84 adaptor subunit and is controlled by Ras and G-betagamma proteins. Ras controls p110g directly and work in mice has revealed the physiological importance of this interaction. In contrast, the roles of p84, p101 and of Gbg remain debated and largely undefined in vivo. A favoured hypothesis, based on in vitro and "in transfecto" data, is that p84/p110g is regulated by Ras and p101/p110g by Gbg.
Our unpublished data from a p84-/- mouse line shows p84 has unique roles in neutrophils. As a result of a collaboration with Roger Williams we can now derive mice expressing a point-mutated, fully-active Gbg-insensitive p110g. These mice, in conjunction with p84-/- x p101-/- and other published lines, will allow us to define how PI3Kg is regulated by Gbg and Ras in vivo and to test the above hypothesis directly.
In the detail of our unpublished data we find that loss of p84, but not p101, abolishes fMLP-stimulated ROS formation, despite very similar impacts on the lipid product of PI3Kg activity, PIP3. We hypothesize this discrepancy results from PI3Kg being found in spatially discrete compartments based on membrane nano-domains. The access of PI3Kg, and hence PIP3, to these nano-domains is determined by adaptor-specific regulation and restricts signalling to similarly targeted PIP3-effectors. We aim to test this idea by visualizing endogenous PI3Kg at high resolution with a range of techniques.

Planned Impact

Who will benefit from our research?
In the shorter term; this would include the academic communities within which we work, however, this will not be discussed further here (described in Academic Beneficiaries). (1) Commercial interests in those fields (this application has built a MICA agreement with GSK, however, both companies with similar interests would also benefit from any publications and presentations (2) The post-holder through the training and experience of working in a world-class research environment. (3) The MRC through delivery of their missions and other funding agencies supporting our work, currently the BBSRC, where this application will serve to increase the impact of experiments that delivered preliminary results presented in these documents. (4) Our institute, BI. In the longer term, (5), the health-care sector, patients and the UKs economy.

How will they benefit?
1) Commercial interests including both our MICA partner and others, will benefit from our publishable results that will yield an integrated understanding of how a currently "hot" target for pharmaceuticals, PI3Kgamma, functions and is regulated in vivo. This will offer alternative approaches to interfering with the pathway, other than ATP-competitive inhibitors favoured currently. It will also test the function of PI3Kgamma and how it is specifically regulated in accepted mouse models of inflammatory disease that are used in the pharmaceutical sector as "proof-of-principle" and hence will engage with their agenda. The commercial sector has, and will, benefit form staff trained in our lab (see below). In the last 5yrs 7 PhD students or PDRAs trained in our lab have found employment in companies because of the relevance of our work to the commercial sector and the skills/talents of the individuals part derived from their training at BI.
2) The post holders. The host labs past PDRAs and PhD students have been very successful in finding further employment in both the academic, commercial and medical charity sectors. This results from the quality and relevance of the training they receive and the contacts we have created.
3) Our work will contribute to the delivery of the MRCs mission (and related medical charities, see above). This arises from; (i) it addressing work that is relevant to "Resilience, repair and replacement" and "Living a long and healthy life" themes within the MRCs first strategic priority. Neutrophils are now thought to be key players in sustaining a number of chronic inflammatory diseases (part of the "tissue disease and degeneration objective") and hence as our work addresses the basic molecular mechanisms underpinning one of the central pro-inflammatory pathways in neutrophils our work maps into this priority. (ii) Furthermore, neutrophils are key players in innate resistance to infection that is also relevant to the "natural protection" objective in this theme. (iii) The relevance of our work to the pharmaceutical industry (evidenced by the MICA agreement with GSK) and the fact that many companies are currently attempting to derive novel anti-inflammatories by making inhibitors of PI3Kgamma shows our work is also relevant to the second MRC strategic priority "Research to people", at many levels.
4) Our work will support BIs mission to become a world-leading research institute addressing "life-long health and well-being" (see above).
5) Our work has, and will, contributed to the UK economy through our collaborations with industry (including the MICA agreement with GSK, here) resulting in the original characterisation of what is now considered a major drug target (PI3Kgamma), contributions to validating the principle that it is a potential drug target and advice and leading contributions on how to assay that enzyme in vivo. This work will develope that history. Furthermore, as PI3Kgamma inhibitors progress through clinical trials this work has a real possibility to impact patient experience/quality of lif

Publications

10 25 50
 
Title Novel mouse models with genetically manipulated PI3K gamma function. 
Description As we planned in the grant we have successfully created and vaildated a set of novel GM mouse lines in which we have knocked-in mutations into the endogenous PI3Kgamma regulatory (p101) and catalytic (p110g) subunits; point mutations that we have shown render them insensitive to G-protein beta gama subunits. We have also generated the first double regulatory subunit knock-out line (p84-/- x p101-/-; this was a non-trivial undertaking as the genes neighbour one another and hence inter breeding the single KO strains is extremyl unlikely to yield homozygous double KOs) . These mice have allowed us to determine the physiological roles of Gbeta gamma subunit binding to PI3Kgamma as distinct to regulation by for example Ras proteins. Hence in total we have created 4 novel lines (plus control strains) ; a p101-point mutant knock-in, p110g-point mutant knock-in, and strains expressing both mutations or lacking (KO) both p84 and p101 regulatory subunits. These unique resources form the basis of a large paper we are in the final stages of preparing, 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? No  
Impact It has enabled us to under take the first full dissection of the roles of Gbeta gamma subunits in regulation of PI3Kgamma in vivo and to establish that Gbeta gamma binding to p101 alone is physiologically important. We are finalising experiments and assembling a major paper with these results at the moment. 
 
Description GSK/Gus 
Organisation GlaxoSmithKline (GSK)
Department Respiratory Biology GSK
Country United Kingdom 
Sector Private 
PI Contribution Research into the physiological roles of Pi3kgamma in inflammatory signalling in neutrophils
Collaborator Contribution Knowledge of commercially important directions and implications of our experiments
Impact In the first year of the study.
Start Year 2011
 
Description Ian Prior 
Organisation University of Liverpool
Department School of Biological Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution we are neutrophil preparations from mice expressing tagged p8/p101 PI3Kgamma regulatory subunits. We will use them to prepare EM grids with ripped plasma membrane sheets for analysis of distribution of p84 v p101 within nano domains by EM
Collaborator Contribution EM analysis and training in analytical methods
Impact The collaboration is multi-disciplinary and involves combined use of mouse genetics, EM microscopy and application of mathematical tools to biological data
Start Year 2010
 
Description Roger Williams 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We are making point mutant mouse knock-ins of PI3kgamma subunits on the basis of structural predictions from work in Roger Williams lab
Collaborator Contribution Structural analysis of PI3K subunits
Impact Not so far on this grant
 
Description Invited lecturer to international meetings (average 2-3 per year) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact promoted discussions, collaborations

scientific collaborations, joint grants and publications
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Schools day 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact School children from across the country attended an event at Babraham
Year(s) Of Engagement Activity 2018
 
Description Work experience for year 12 school students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Work shadowing for a yr 12 school student to understand biomedical research

Student applied and got an offer on a biomedical degree course
Year(s) Of Engagement Activity 2014