Exo-Net

Lead Research Organisation: University of Liverpool
Department Name: Biomedical Sciences

Abstract

In order to survive human cells must continuously release and internalize both soluble and membrane bound factors. The most common method by which cells release factors such as serum proteins, peptide hormones and antibodies is called ?exocytosis?. In human cells exocytosis can happen in three ways. Every cell in our body is constantly producing small vesicles, which fuse to the plasma membrane. This is how we replenish the external membrane around our cells. In addition some specialized cells like neurons and secretory cells posses a population of specialized vesicles or granules, which only fuse with the plasma membrane when the cell is stimulated. It is very important that this form of exocytosis is tightly controlled as neurons must only transmit signals at the appropriate time, and the level of molecules like peptide hormones released into the circulation must be carefully controlled. Because exocytosis is an essential process, it is not surprising that defects in this process can result in different forms of human diseases including neuronal and immunological disorders, reproductive problems and diabetes. Although we now have a fairly good understanding of how exocytosis works in human cells there are still many things that we do not understand. To increase our understanding of this important process we plan to use a range of complementary technologies to build maps that show which proteins interact at different stages of exocytosis. We also intend to use new techniques to study these interactions in living cells. By building these ?living maps? we will have a much better understanding of how exocytosis is controlled in different human cells. This information could then help us to develop rational strategies for regulating the release of factors from specific cell in the body.

Technical Summary

Exocytosis is an essential, conserved process that occurs in all eukaryotic cells. In general terms three forms of exocytosis occur in human cells: Constitutive exocytosis, which involves the movement of vesicles from the trans-Golgi network to the plasma membrane. Regulated exocytosis, which involves the controlled release of preformed vesicles or secretory granules and a variation of regulated exocytosis, in which lysosome or endosome derived organelles fuse with the plasma membrane. Given the absolute requirement for exocytosis in human cells it is not surprising that defects in this process lead to a broad range of human diseases including: neuronal and immunological disorders, reproductive problems, albinism and diabetes. In recent years there has been a significant increase in our understanding of the fundamental mechanism of exocytosis in mammalian cells and the proteins that are required. However, there are still many unanswered questions. Although the core components of the exocytic machinery have been identified we still know very little about how the different steps of exocytosis are regulated, or the spatial, kinetic or conditional aspects of many protein-protein interactions. In this project we aim to address these important issues by using complementary bioinformatic and high throughput proteomic techniques to identify novel pathway components and define the full extent of pathway connectivity. In addition we propose to use live cell protein interaction assays to investigate the conditional spatial and temporal aspects of selected protein-protein interactions. To maximize the impact of our studies we have analyzed 90 human proteins, which are known to be involved in exocytosis. For each of these proteins we have identified all known interaction partners, the method used to define each interaction, and any known interaction partners for homologues of these proteins in other species (interlogs). By studying this data and analyzing how these interactions fit together within a graphical network we were able to identify proteins for which little interaction data is known, areas of the network which are fragmented or unconnected and many potential interlogs that have not yet been investigated in mammalian cells. By focusing our research on these candidate proteins we will be able to generate a far more comprehensive picture of exocytosis in human cells. This information will provide a novel insight into the mechanism of exocytosis in human cells and drive forward future functional studies.

Publications

10 25 50
 
Description Neuro-Net
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Strategies for mapping the Human interactome
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guidance committee
 
Description Non-clinical PhD Studentships
Amount £150,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2014
 
Description North West Cancer Research
Amount £36,000 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2009 
End 05/2010
 
Description PhD studentship
Amount £146,279 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description Research Grant (CO-PI)
Amount £1,974,893 (GBP)
Funding ID BSR10882 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2008 
End 03/2014
 
Description Small Capacity Building Grant
Amount £19,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2012 
End 12/2013
 
Description Systems Biology Studentship PTG
Amount £290,160 (GBP)
Funding ID BBF5290031 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 10/2013
 
Description The North West Cancer Research Bioinformatics Fellowship
Amount £185,000 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description Wellcome PhD student
Amount £75,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description Wellcome Trust DTG
Amount £150,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 10/2016
 
Description Wellcome Trust DTG
Amount £150,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 10/2016
 
Title Human E2/E3-RING interaction networks 
Description The first high density binary protein interaction map of human ubiquitin E2-E3 RING proteins. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Protein ubiquitination is a key regulatory mechanism in human cells understanding the partner preferences within this system is fundamental to many physiological and pathological processes. The map will provide a guide to future rational intervention strategies. 
 
Title Human E3-RING / E3-RING interaction database 
Description The first database of homo and heterotypic E3-RING interactions. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact widely accessed dataset: Highlighted the EBIs dataset of the month when published. 
 
Title Reagents for investigationg ubiquitination in human cells 
Description a collection of wild type and mutant forms of all human E2 ubiquitin conjugating enzymes and ~180 E3 RING proteins 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Protein ubiquitination is involved in almost every process within human cells. these reagents will be valuable to many areas of research. 
 
Title high throughput screening of binary protein interactions 
Description When working at the MRC HGMP-RC we developed reagents and procedures to perform high-throughput yeast two hybrid screens which were offered as a collaborative service. This facilitated and advanced the work of several other UK research groups. Screens performed by our group led directly to publications in EMBO, Current Biology and Cell. When the HGMP-RC was closed down we continued to receive requests for assistance with yeast two-hybrid studies. we have tried to help these groups where possible and we are still involved in active collaborations with several groups at the CIMR and LMB who continue to request help. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Screens performed by our group led directly to publications in EMBO, Current Biology and Cell. 
 
Description Development of computational tools to study human disease networks 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution development of computational tools to interpret and investigate human protein interaction pathways
Collaborator Contribution Provision of disease related patient data which was used to develop computational algorithms used to predict novel componenets of both physiological and pathological pathways.
Impact Thwo manuscripts relating to this collaborative work are in preparation
Start Year 2008
 
Description Network analysis of quantitative proteomic data 
Organisation University of Liverpool
Department School of Biological Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided expertise in the generation and analysis of complex protein interaction networks which was essential for data interpretation in a large scale proteomic study
Collaborator Contribution Access to quantitative proteomic expertise and facilities
Impact One publication 26222723 was generated as a result of this publication. Collaboration was multi-disciplinary in that we provided computational expertise to a wet lab project.
Start Year 2007
 
Description Physical and bioinformatic analysis of protein interactions 
Organisation Dana-Farber Cancer Institute
Department Center for Cancer Systems Biology (CCSB)
Country United States 
Sector Academic/University 
PI Contribution Since 2005 my research team have been developing a map of numan E2 Ubiquitin protein interactions. This work has been almost totaly funded through MRC studentships. However, technologies and most importantly computational algorithma developed to aid the Exo-Net project have provided invaluable tools for a broad range of in house and collaborative research projects
Collaborator Contribution Protein interactions detected in our yeast two-hybrid library screens were further investigated leading to new insights into the molecular mechanisms of endocytosis and viral immunevasion strategies.Researchers at the CRG Systems Biology unit performed computational predictions which were correlated with our protein interaction data. Further computational and experimental correlations are ongoing.Contribution of human cDNA clones from human orfeome collection.Performed invitro protein ubiquitination assays which were then correlated with our binary protein interaction data to increase confidence in human protein interaction networks.
Impact MRC funded projects have facilitated the production of very high quality libraries and highthroughput screening technologies which have driven many active research projects. We continue to make these resourses available to a large number on UK and international researchers. As such the impact and broad utility of these resourses has been very significant. Given the relevance of these reagents to many areas of medical research I fully expect their utility to continue for many years to come. Todate they have led to at laest 6 peer reviewed colaborative publications (main:19549727, others:16339213, 16601694, 16730941, 17398095, 17711858, 18775314) involving world leading groups from the University of Cambridge, the LMB, Harvard and the EMBL Systems Biology Centre in Barcelona.
 
Description Physical and bioinformatic analysis of protein interactions 
Organisation European Molecular Biology Laboratory
Country Germany 
Sector Public 
PI Contribution Since 2005 my research team have been developing a map of numan E2 Ubiquitin protein interactions. This work has been almost totaly funded through MRC studentships. However, technologies and most importantly computational algorithma developed to aid the Exo-Net project have provided invaluable tools for a broad range of in house and collaborative research projects
Collaborator Contribution Protein interactions detected in our yeast two-hybrid library screens were further investigated leading to new insights into the molecular mechanisms of endocytosis and viral immunevasion strategies.Researchers at the CRG Systems Biology unit performed computational predictions which were correlated with our protein interaction data. Further computational and experimental correlations are ongoing.Contribution of human cDNA clones from human orfeome collection.Performed invitro protein ubiquitination assays which were then correlated with our binary protein interaction data to increase confidence in human protein interaction networks.
Impact MRC funded projects have facilitated the production of very high quality libraries and highthroughput screening technologies which have driven many active research projects. We continue to make these resourses available to a large number on UK and international researchers. As such the impact and broad utility of these resourses has been very significant. Given the relevance of these reagents to many areas of medical research I fully expect their utility to continue for many years to come. Todate they have led to at laest 6 peer reviewed colaborative publications (main:19549727, others:16339213, 16601694, 16730941, 17398095, 17711858, 18775314) involving world leading groups from the University of Cambridge, the LMB, Harvard and the EMBL Systems Biology Centre in Barcelona.
 
Description Physical and bioinformatic analysis of protein interactions 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Since 2005 my research team have been developing a map of numan E2 Ubiquitin protein interactions. This work has been almost totaly funded through MRC studentships. However, technologies and most importantly computational algorithma developed to aid the Exo-Net project have provided invaluable tools for a broad range of in house and collaborative research projects
Collaborator Contribution Protein interactions detected in our yeast two-hybrid library screens were further investigated leading to new insights into the molecular mechanisms of endocytosis and viral immunevasion strategies.Researchers at the CRG Systems Biology unit performed computational predictions which were correlated with our protein interaction data. Further computational and experimental correlations are ongoing.Contribution of human cDNA clones from human orfeome collection.Performed invitro protein ubiquitination assays which were then correlated with our binary protein interaction data to increase confidence in human protein interaction networks.
Impact MRC funded projects have facilitated the production of very high quality libraries and highthroughput screening technologies which have driven many active research projects. We continue to make these resourses available to a large number on UK and international researchers. As such the impact and broad utility of these resourses has been very significant. Given the relevance of these reagents to many areas of medical research I fully expect their utility to continue for many years to come. Todate they have led to at laest 6 peer reviewed colaborative publications (main:19549727, others:16339213, 16601694, 16730941, 17398095, 17711858, 18775314) involving world leading groups from the University of Cambridge, the LMB, Harvard and the EMBL Systems Biology Centre in Barcelona.
 
Description Physical and bioinformatic analysis of protein interactions 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Since 2005 my research team have been developing a map of numan E2 Ubiquitin protein interactions. This work has been almost totaly funded through MRC studentships. However, technologies and most importantly computational algorithma developed to aid the Exo-Net project have provided invaluable tools for a broad range of in house and collaborative research projects
Collaborator Contribution Protein interactions detected in our yeast two-hybrid library screens were further investigated leading to new insights into the molecular mechanisms of endocytosis and viral immunevasion strategies.Researchers at the CRG Systems Biology unit performed computational predictions which were correlated with our protein interaction data. Further computational and experimental correlations are ongoing.Contribution of human cDNA clones from human orfeome collection.Performed invitro protein ubiquitination assays which were then correlated with our binary protein interaction data to increase confidence in human protein interaction networks.
Impact MRC funded projects have facilitated the production of very high quality libraries and highthroughput screening technologies which have driven many active research projects. We continue to make these resourses available to a large number on UK and international researchers. As such the impact and broad utility of these resourses has been very significant. Given the relevance of these reagents to many areas of medical research I fully expect their utility to continue for many years to come. Todate they have led to at laest 6 peer reviewed colaborative publications (main:19549727, others:16339213, 16601694, 16730941, 17398095, 17711858, 18775314) involving world leading groups from the University of Cambridge, the LMB, Harvard and the EMBL Systems Biology Centre in Barcelona.
 
Description Cold Spring Harbor presentation (poster) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Exo-Net results and novel computational network analysis methods were presented as part of the international interactome/Systems Bioloby meeting.

Requests for collaboration were recieved and these are now ongoing.
Year(s) Of Engagement Activity 2009
 
Description Course organiser/ Instructor 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact for each of the past four years 16 postgraduate and/or postdoctoral researchers have attend a one week course held in Hinxton Cambridge. During this time they were trained in a broad range of techniques which can be used to study protein-protein interactions.

Several course participants have successfully used techniques to advance research projects and start up independent research projects
Year(s) Of Engagement Activity 2009,2010,2011,2012
 
Description Human Interactome strategy think tank meeting Cold Spring Harbor 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact A group of ~22 scientists were invited to Cold Spring Harbour to discus strategies for mapping the human interactome. I was one of Two UK scientists invited to attend

This lead to a subsequent invitation to present at the next international Human interactome meeting held in Cambridge. Attendance at this meeting also established our group as the leading yeast two-hybrid/network biology group in the UK within the UK.
Year(s) Of Engagement Activity 2007
 
Description Speaker at third anual international Human Interactome Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Selected presentation covering all ongoing network biology projects from my group. This included detailed description of a large human ubioquitination network assembled by my MRC funded PhD student (Gabriel Markson). And a description of the practical and bioinformatic studies in progress on the Exo-Net project.

Presentation at two out of the last three international human interactome meetings has resulted in the development of key strategic collaborations which are assisting our ongoing projects. In addition these collaborations will continue to underpin future applications for funding and provide access to important reagent sets. In particular we have established an ongoing active collaboration with two groups at the Dana-Faber Cancer institute in Harvard, which has provided access to the human orfeome clone set. We are also arranging for students to go to Harvard to generate a human splice variant resource which we will use to investigate differential interaction patterns between protein isoforms.
Year(s) Of Engagement Activity 2007
 
Description UK JAPAN Collaborative network: Experimental and computational analysis of human protein interaction networks 
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 Approximately 30 people were involved in a UK/Japan collaborative meeting

New international collaborative projects are being established
Year(s) Of Engagement Activity 2011
 
Description Widening Participation day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact approximately 35 students attended a lab open day to increase numbers of local students applying to study medical science at University

Very positive student feedback. Further open days have been arranged to increase widening participation.
Year(s) Of Engagement Activity 2013