Proteomics to identify signalling in macrophage and phagosome biology

Lead Research Organisation: University of Dundee

Abstract

Phagocytosis is an important mechanism of specialised immune cells such as macrophages helping to fight of infections. However, many pathogens are able to subvert phagosome functions and are able to survive within macrophages, most prominently Mycobacterium tuberculosis, the causative agent of tuberculosis. Therefore, it is of great importance to understand which signaling events regulate phagosome biogenesis and how pathogens undermine it. My group will focus on identifying novel signaling pathways on the phagosome using proteomics, a mass spectrometry-based large-scale technique that allows us to identify thousands of proteins and protein modification in a single experiment. In addition, we will try to identify how a specific pathogen factor of Mycobacterium tuberculosis changes signaling on the phagosome leading to the survival of the bacteria in an otherwise hostile environment within macrophages. These results might help us in the future to understand the molecular mechanism of phagosome subversion by pathogens.

Technical Summary

Aims 1. Characterisation of protein phosphorylation events modulating phagosome biogenesis. 2. Characterisation of host-targets of bacterial pathogen factors modulating phagosome biogenesis and other immune functions in macrophages. Plan of Investigation Phagosomes are organelles formed by the uptake of particles or pathogens by specialised immune cells such as macrophages. Phagosomes mature by a number of fusion events with endosomes and the lysosome, forming a phagolysosome in which these pathogens are killed and degraded. Antigens of the microbes are then presented via the MHC class I and II pathways, thus linking innate and adaptive immunity. Unfortunately, many pathogens including Mycobacterium tuberculosis, the causative agent of tuberculosis, are able to subvert phagosome maturation/biogenesis by injection of pathogenic factors into the phagosome, enabling them to survive. Very little is known about the signaling events leading to phagosome biogenesis. We will analyse the proteome and phosphoproteome of phagosomes by large-scale mass spectrometry approaches. For this, we will generate high purity phagosome fractions from mouse macrophage cell cultures by the use of opsonised polystyrene beads and subsequent sucrose gradient ultra centrifugation. By analysing phagosomes internalised upon different routes of entry and different maturation times, we will generate a large dataset that will help us understand the signaling events regulating phagosome function over the whole time-scale important for phagosome function. In addition, we will analyse the phagosome proteome and phosphoproteome upon challenge with secreted bacterial effectors such as the mycobacterial protein kinase G which has been shown to be involved in blocking phagosome maturation, enabling Mycobacterium tuberculosis to survive within the phagosome of macrophages. As phagocytosis is a key mechanism in innate and adaptive immunity, identification of the mechanisms of phagosome biogenesis will be of importance for our understanding of infectious diseases and vaccine design. Moreover, insights of how pathogens subvert phagosome maturation will enable us to target bacterial pathogen factors for drug design.

Publications

10 25 50

publication icon
Gandin V (2016) mTORC1 and CK2 coordinate ternary and eIF4F complex assembly. in Nature communications

 
Description (Magicbullet Reloaded) - Small-Molecule Drug Conjugates for Targeted Delivery in Tumor Therapy
Amount € 3,915,902 (EUR)
Funding ID 861316 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2019 
End 10/2023
 
Description A multi-application microarray robot for agri-food research, industrial biotechnology and on-chip manufacturing
Amount £334,656 (GBP)
Funding ID BB/S019804/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 06/2020
 
Description BBSRC CASE studentship
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 08/2020
 
Description BBSRC CASE studentship
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2016 
End 01/2020
 
Description Deciphering the role of phagosomal ubiquitylation in innate immunity
Amount £1,832,057 (GBP)
Funding ID 215542/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 09/2024
 
Description EPSRC Centre for Doctoral Training in Molecular Sciences for Medicine
Amount £7,291,056 (GBP)
Funding ID EP/S022791/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 10/2027
 
Description MJFF LEAPS Team Grant
Amount $232,956 (USD)
Organisation Michael J Fox Foundation 
Sector Charity/Non Profit
Country United States
Start 01/2013 
End 12/2015
 
Description MRC DGF
Amount £284,483 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 09/2011 
End 08/2014
 
Description Targeting a new kingdom: the nature and significance of Type VI secretion system-mediated anti-fungal activity
Amount £2,729,187 (GBP)
Funding ID 215599/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2019 
End 11/2024
 
Description University of Dundee and Wellcome Trust Translational Medical Research Fund
Amount £10,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 09/2014
 
Description Utilising the MALDI-TOF E3-ligase assay to measure quantitative ubiquitination kinetics and mechanistic profiling of PROTACs
Amount £100,279 (GBP)
Funding ID BB/T508378/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2023
 
Description Wellcome Trust Multi-User Equipment Grant
Amount £578,689 (GBP)
Funding ID 212947/Z/18/Z 
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 12/2018 
End 11/2023
 
Title Development of a MALDI-TOF Deubiquitylase assay for drug discovery 
Description This assay allows the high-throughput analysis of chain specificity of Deubiquitylases (DUBs) and also allows a drug discovery screen of DUBs against many compounds. Because it uses more physiological substrates than the current fluorescence-based assays, it provides less false-positives. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact We have several collaborations with pharmaceutical companies to test their compounds. We identified the first highly-specific DUB inhibitor with one of the companies and plan to publish this soon. This assay allows pharma companies to target DUBs that are dysregulated in diseases such as cancer. It is likely that this assay will generate substantial income to the MRC UNit by providing a service to pharma companies. 
 
Title Development of hydrophilic Strong-Anion Exchange Chromatography 
Description We developed in collaboration with Thermo-Fisher Scientific a novel hydrophilic strong-anion exchange chromatography (hSAX) which enabled us to identify very large numbers of proteins in cell lysates by mass spectrometry. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Enables many other groups to perform large scale proteomics. 
 
Title Rabs as target for PINK1 
Description Together with Miratul Muqit (MRC PPU), we identified that PINK1, a kinase mutated in Parkinson's disease, is inducing phosphorylation of Rab GTPases (Lai et al, EMBO J, 2015). 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2015 
Provided To Others? Yes  
Impact This is a new read-out of PINK1 activity that could be used for drug discovery in the Parkinson's disease field. 
 
Title The MALDI TOF E2/E3 ligase assay 
Description This is the first high-throughput label-free assay for E2/E3 ligases. For the HOIP complex, which generates linear ubiquitin chains and which is an attractive drug target, it is the first high-throughput assay overall. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This will have an impact on drug discovery for E2/E3 ligases. Once it is published we will make it available to the research community. 
 
Description Developing high-throughput MALDI TOF mass spectrometry for Drug Discovery 
Organisation Bruker Corporation
Department Bruker Daltonik GmbH
Country Germany 
Sector Private 
PI Contribution We develop new enzymatic assays for high throughput MALDI TOF mass spectrometry. We also improve and develop existing workflows.
Collaborator Contribution Bruker provides prototype instrument access, prototype materials/consumables, software development.
Impact We have obtained a BBSRC CASE studentship together. We have now integrated a working relationship with several pharma companies including AstraZeneca, GSK and Merck.
Start Year 2015
 
Description Development of novel liquid chromatography for large-scale proteomics 
Organisation Thermo Fisher Scientific
Country United States 
Sector Private 
PI Contribution Development of novel chromatographic techniques for large-scale proteomics and phosphoproteomics.
Collaborator Contribution Provided new materials, expertise and consumables.
Impact one publication in Journal of Proteome Research (Ritorto et al, 2013; PMID: 23294059), one publication in Bioanalysis (Ritorto et al, 2013; PMID: 24053235). A nice collaboration with industry partners at Thermo-Fisher Scientific, their chemists and our biochemical and cell biological expertise. Applied together for a BBSRC CASE studentship in 2014.
Start Year 2011
 
Description Drug Discovery in the ubiquitin system 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Utilising high-throughput MALDI TOF mass spectrometry for drug discovery.
Collaborator Contribution compounds and expertise.
Impact We received a BBSRC iCASE studentship.
Start Year 2019
 
Description Identification of LRRK2 substrates by mass spectrometry 
Organisation Dana-Farber Cancer Institute
Country United States 
Sector Hospitals 
PI Contribution Proteome and phosphoproteome analysis of LRRK2 mutant strains. Various tissues.
Collaborator Contribution Biochemical validation by Dario Alessi's group.
Impact Multidisciplinary: Biochemistry (Dario Alessi group), Medicine (Nicholas Wood group), Chemists (Nathanael Gray group), Companies (GSK, Merck) Published paper in ELife.
Start Year 2011
 
Description Identification of LRRK2 substrates by mass spectrometry 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Proteome and phosphoproteome analysis of LRRK2 mutant strains. Various tissues.
Collaborator Contribution Biochemical validation by Dario Alessi's group.
Impact Multidisciplinary: Biochemistry (Dario Alessi group), Medicine (Nicholas Wood group), Chemists (Nathanael Gray group), Companies (GSK, Merck) Published paper in ELife.
Start Year 2011
 
Description Identification of LRRK2 substrates by mass spectrometry 
Organisation Max Planck Society
Country Germany 
Sector Charity/Non Profit 
PI Contribution Proteome and phosphoproteome analysis of LRRK2 mutant strains. Various tissues.
Collaborator Contribution Biochemical validation by Dario Alessi's group.
Impact Multidisciplinary: Biochemistry (Dario Alessi group), Medicine (Nicholas Wood group), Chemists (Nathanael Gray group), Companies (GSK, Merck) Published paper in ELife.
Start Year 2011
 
Description Identification of LRRK2 substrates by mass spectrometry 
Organisation University of Dundee
Department MRC Protein Phosphorylation and Ubiquitylation Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Proteome and phosphoproteome analysis of LRRK2 mutant strains. Various tissues.
Collaborator Contribution Biochemical validation by Dario Alessi's group.
Impact Multidisciplinary: Biochemistry (Dario Alessi group), Medicine (Nicholas Wood group), Chemists (Nathanael Gray group), Companies (GSK, Merck) Published paper in ELife.
Start Year 2011
 
Description Legionella phagosomes 
Organisation Charité - University of Medicine Berlin
Country Germany 
Sector Academic/University 
PI Contribution Proteome analysis of Legionella containing phagosomes. Bioinformatic analysis.
Collaborator Contribution IFN-beta depending changes on the phagosome. mechanistic analyses
Impact Multidiscilinary with medical immunologists from Charite, Berlin and TU Munich. Publication in PLOS Pathogens (Naujoks et al, PMID: 26829557)
Start Year 2012
 
Description Proteome analysis to characterise PINK-1 signalling 
Organisation University of Dundee
Department MRC Protein Phosphorylation and Ubiquitylation Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution proteome analysis of PINK-1 wt or kinase-dead cells.
Collaborator Contribution Biochemistry, validation by Miratul Muqit's group.
Impact Kazlauskaite et al, Biochem J (PMID:24660806) Kazlauskaite et al, EMBO Rep (PMID:26116755 ) Lai et al, EMBO J (PMID: 26471730 ) patent shared by Miratul Muqit and Matthias Trost
Start Year 2011
 
Description Proteomic characterisation of macrophage signalling 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Proteome analysis of macrophages responding to various stimuli.
Collaborator Contribution Simon Arthur's lab performs all the biochemistry, validation.
Impact manuscript in preparation.
Start Year 2012
 
Description Synaptosome Proteomics 
Organisation University of Edinburgh
Department Research Institute for Digital Communications
Country United Kingdom 
Sector Academic/University 
PI Contribution We analysed synaptosomes by proteomics generated by the collaborator's lab of Mike Cousin at University of Edinburgh.
Collaborator Contribution This is the partner's main biological focus and expertise. they provided synaptosome and endosome samples. we provide the proteomics.
Impact several papers in preparation.
Start Year 2013
 
Description T6SS in Serratia marcescens 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Proteome analysis of secretome of Serratia marcescens,
Collaborator Contribution Functional analysis of type-6 secretion system of Serratia marcescens.
Impact Multi-disciplinary (Microbiology, Stuctural Biology, Proteomics) First publication in Mol Cell Proteomics, Fritsch MJ et al, 2013 two publications planned for 2016.
Start Year 2011
 
Description The draft human organellar proteome 
Organisation University of Cambridge
Department Cambridge Centre for Proteomics
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab provides expertise in large-scale proteomics and organellar purification.
Collaborator Contribution The Lilley lab provides similar but complimentary expertise and bioinformatics expertise.
Impact none yet as it only started in 2014.
Start Year 2014
 
Description Debating Matters (Fife) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Judge at Debating Matters or the Fife/Stirling qualifying rounds


Judge at Debating Matters or the Fife/Stirling qualifying rounds
Year(s) Of Engagement Activity 2014
 
Description Debating Matters Judge 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Due to my position was asked to become Debating Matters Judge, judging pupils' performance during debates.
http://www.debatingmatters.com/people/matthias_trost/

Made public aware of MRC Unit's success in science.
Year(s) Of Engagement Activity 2012,2013
 
Description Interview about PINK1 Substrate Findings - The Times (2015) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Drs. Muqit and Trost were interviewed by Mike Wade at The Times about their recent findings about a PINK1 substrate and its implications for Parkinson's disease therapeutic development.

These most recent findings are quite critical to a more comprehensive understanding of PINK1 function and its implications in Parkinson's pathobiology. The broader public, including patients, have engaged with MRC PPU researchers to better understand its implications.
Year(s) Of Engagement Activity 2015
 
Description Interview for CLSPA career pathways project 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Gave interview with professional TV team. The purpose of the project is to give a view of the very different career paths, and the choices that influenced them along the way, which have been followed by staff who have held a Post Doctoral position at some point in their lives. Through a series of 'career timelines', portraits and interviews we will produce a resource which will be as engaging as it is informative for PhD students and Post Docs as well as the general public.

no feedback yet.
Year(s) Of Engagement Activity 2012,2013
 
Description MRC-PPU Collaboration with Baldragon Academy 2014-2015 
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 The Medical Research Council's Protein Phosphorylation Ubiquitylation Unit (MRCPPU), part of The University of Dundee, has prioritized public engagement in an effort to engage the general public and ensure that the research activities and breakthroughs are communicated to the community. Of equal importance in these communication efforts is educational outreach to students within the Dundee community.

Thus, during session 2014 -2015 and 2015-2016 school year, the MRC-PPU will partner with a local secondary school -- Baldragon Academy (BA). Teachers in BA's Science Department will collaborate with scientists at the MRC-PPU in an educational outreach effort (see Appendix 1). The purpose of this project is to increase interest and engagement in science and related careers. It will be starting in August 2014 with the S1 pupils.

Scientists from the unit will be working with the pupils on a monthly basis at the school during their science classes and will be providing them with opportunities to take part in various science experiments and demonstrations (aligned with Scotland's Curriculum for Excellence). The scientists are leaders in their field of research and as such come from all over the world. They are currently based in Dundee.

Thus far, student have been very enthusiastic about the labs and very receptive to the volunteers. They have asked a multitude of questions and have even asked volunteers back to visit. We have also had numerous students of different ages from the school ask to participate in work experience activities to learn more about the Unit and science in general.
Year(s) Of Engagement Activity 2014,2015
 
Description Open Day Parkinson's Disease 2013 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Our objective with this event was to communicate our research to members of the public. We advertised our event through local Parkinson's Disease patient groups and charities. 20 visitors attended including patients, relatives of patients, charity workers and school children.

Feedback from the visitors was positive.
100% of guests "completely enjoyed the event.
There was equivalent interest in:
- learning more about cancer research
- meeting and chatting to scientists
- finding out how medical research affects health
100% wanted to attend a similar event in the future.
Year(s) Of Engagement Activity 2013
 
Description Open day Cancer 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Our objective with this event was to communicate our research to members of the public. We advertised our event through local cancer patient groups and cancer charities. 40 visitors attended including cancer survivors, current cancer patients, relatives of cancer patients, cancer charity workers and school children.

The day started with a general introduction to our unit and cancer research in our unit from Prof John Rouse. Prof Rouse, Dr Victoria Cowling and Dr Ian Ganley then explained their current research questions. This session was directly followed by a lively question and answer session, including discussion of our research and cancer research in general. During a refreshment break there was opportunity for more informal discussions with the Prof Rouse, Dr Cowling and Dr Ganley. At this time, lab tours were also conducted including demonstrating mass spectrometry, live cell imaging and the MRC-PPU research area. Dr Matthias Trost, Dr Thimo Kurz and Dr Yogesh Kaluthu assisted with the tours. A number of postdocs and PhD students were also involved throughout the day.


Feedback from the visitors was positive.
100% of guests "completely enjoyed the event.
There was equivalent interest in:
- learning more about cancer research
- meeting and chatting to scientists
- finding out how medical research affects health
100% wanted to attend a similar event in the future.
Year(s) Of Engagement Activity 2013
 
Description Our Foreign Skies 
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 Met the public and discussed during the Our Foreign Skies Photography Exhibition at The Mills Observatory
Year(s) Of Engagement Activity 2014
 
Description Oxford talk 2011 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited talk at Oxford University: Systems Biology of the Phagosome, 12. April 2011

Potential collaborations
Year(s) Of Engagement Activity 2011
 
Description Pfizer Talk 2012 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited talk at Pfizer Inc, Proteomics and phosphoproteomics tools to understand human disease, Pfizer, Boston, 13. September 2012

improved collaboration within DSTT.
Year(s) Of Engagement Activity 2012
 
Description Science Week at Newport Primary School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Newport primary hosted a science week for its pupils from the 17th to the 21st of March, 2014. During this week, Deena Leslie Pedrioli (DGEM), Patrick Pedrioli and Thimo Kurz (MRC-PPU), three PIs from the College of Life Sciences in Dundee, helped the children experience first hand what it is like to work in a biology lab. The pupils had the opportunity to peer through the lens of a microscope, to simulate how microbes are transferred, to learn to see objects too small to be seen, and to build cells out of Play-Doh. Students from the College also explained to the pupils what it is like to be a scientist and will answer their questions on the life in the lab.


Many questions were asked by the students. Both students and teachers alike enjoyed the demonstrations.
Year(s) Of Engagement Activity 2014
 
Description Scottish Crucible Panel 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The scheme enabled 30 talented researchers from a variety of disciplines to come together to explore and expand their creative capacity and problem-solving potential in ways they may never have considered before.


This event provided academicians from very diverse backgrounds with the opportunity to meet and discuss topics of common interest and learn of each others' perspectives.
Year(s) Of Engagement Activity 2014
URL http://www1.hw.ac.uk/scottishcrucible/