Mechanisms of transmembrane signalling by tetraspanins

Lead Research Organisation: University of Birmingham
Department Name: Clinical and Experimental Medicine

Abstract

In this project the structures and interactions of the human tetraspanin proteins will be investigated by heteronuclear magnetic resonance spectroscopy. We have discovered that tetraspanin C-termini are bound by the tandem PDZ proteins of syntenin, and intend to elucidate the structural basis of these interactions in order to understand how they influence receptor signaling and endocytosis. The novel regulatory effects of PDZ domain phosphorylation and stabilizing terminal extensions will be investigated in order to build a model of the signaling mechanism. The extracellular domain has been expressed for NMR analysis of its lipid interaction sites and protein docking studies, providing a basis for defining its function as an cell surface receptor. Tetraspanins have been expressed as functionally intact full length proteins, and production will be scaled up to analyse their structural and binding properties in vitro. In particular, we seek to determine the oligomeric state and structural properties of the proteins in the free and ligand bound states using mixed micelles to solubilize the intact receptor. Together with collaborative in vivo cell biological studies this will provide a better understanding of the structural mechanism of tetraspanin signaling.

Technical Summary

Tetraspanins are an extensive family of under-characterized integral membrane proteins. Each family member contains four transmembrane regions, two extracellular domains, and variable cytoplasmic termini. Their total size is 23 ? 40 kDa. These structural domains determine the specific interactions of the tetraspanin with ligand proteins including immune receptors and viral envelope proteins, as well as with phospholipids, glycolipids and cholesterol within transmembrane-enriched microdomains. The structural basis of these interactions is unclear, limiting our mechanistic understanding of tetraspanin functions and precluding rational drug design.

Tetraspanins are found in mammals, plants, fungi and protozoa. Humans contain 33 tetraspanins, which are generally involved in cell adhesion, migration and fusion, and cellular activation and signaling. They are found in essentially all tissues, with each cell type expressing several tetraspanins. Biological roles of mammalian tetraspanins include contributions to kidney, skin and platelet function, sperm?egg fusion, nervous system development, monocyte fusion and immune cell proliferation, although their molecular mechanisms remain poorly understood.

In this project the structures and interactions of the human tetraspanins will be investigated by heteronuclear magnetic resonance spectroscopy. We have discovered that tetraspanin C-termini are directly and specifically bound by tandem PDZ proteins, and intend to elucidate the structural basis of these interactions in order to understand how they influence receptor signaling and endocytosis. The novel regulatory effects of PDZ domain phosphorylation and stabilizing terminal extensions will be investigated in order to build a model of the signaling mechanism. The extracellular domain has been expressed for NMR analysis of its lipid interaction sites and protein docking studies, providing a basis for defining its function as a cell surface receptor. Finally two tetraspanins have been expressed as functionally intact full length proteins, and production will be scaled up to analyse their structural and binding properties in vitro. In particular, we seek to determine the oligomeric state and structural properties of the proteins in the free and ligand bound states using mixed micelles to solubilize the intact receptor. Together with collaborative in vivo cell localization and endocytic studies this will provide a comprehensive structural mechanism of tetraspanin signaling.

Publications

10 25 50
 
Description Biomolecular NMR Infrastructure in the UK Report for MRC and STFC
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact support to develop a network of state-of-the-art regional NMR facilities to keep the UK research infrastructure competitive
 
Description Science Capital Innovative Healthcare Meeting
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact Overduin organised a meeting of industry, NHS and business leaders in Birmingham to support innovation and improve the economics of healthcare. Speakers including Dr. Alan Davies, Chief Medical Officer of General Electric Healthcare argued that ring fencing the NHS budget makes political, not business sense. It was argued that the economics of drug development also need a rethink. The industry has switched to developing targeted therapies, but the high costs involved in the UK are blocking approvals and investments in clinical trials before the true patient benefits are clear, according to Philip Johnson, Professor of Oncology at the University of Birmingham. See www.sciencecapital.org for details.
URL http://www.sciencecapital.co.uk
 
Description MRC DTA Training Programme
Amount £60,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2013
 
Title SMALP system 
Description The styrene maleic acid lipid particle (SMALP) system has been developed to directly solubilize tetraspanins and other membrane proteins from mammalian cells. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Patent: submitted Publication: in JACS in 2009 Collaboration agreements: with MRC-LMB and several pharmaceutical companies for preparation and analysis of membrane protein targets 
 
Title Soluble tetraspanin proteins 
Description Soluble extracellular loop domains from 6 different human tetraspanins that were not amenable to detailed functional studies were produced. Functionality of the recombinant proteins were ensured by their expression and purification from mammalian cells. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact The soluble domains were successfully used to raise polyclonal antibodies against specific tetraspanin proteins. This is providing us with a clear handle to investigate the detailed biochemical and functional characterisation of signalling mediated by these proteins which was not possible earlier. Important insights into the roles of some of these proteins can be analysed in tissue samples and cell lines using these reagents. 
 
Description Analysis of claudin proteins 
Organisation University of Birmingham
Department College of Medical and Dental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Production of claudin proteins as ligands and homologues of tetraspanins including CD81.
Collaborator Contribution Provision of assays for functional activity of claudin proteins
Impact Collaborative paper has been published: Jamshad, M, S Rajesh, Z Stamatakic, JA McKeating, T Dafforn, M Overduin, RM Bill (2008) Structural characterization of recombinant human CD81 produced in Pichia pastoris, Protein Expr Purif. 57:206-16. A PhD studentship has been recruited to further develop this new collaboration and set of claudin targets, which are structurally and functionally related to the original tetraspanin targets.
Start Year 2008
 
Description NMR analysis of signaling targets 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We produced expression constructs and test purification of claudin proteins from E coli and mammalian cells.
Collaborator Contribution We have obtained access to inhibitors, high throughput crystallization and ligand screening methods, and have submitted joint grant applications.Provision of claudin constructs, ligands and binding assays; development of antibodies
Impact A PhD studentship has been recruited to further develop this new collaboration and set of claudin targets, which are structurally and functionally related to the original tetraspanin targets.
Start Year 2009
 
Description NMR analysis of signaling targets 
Organisation University of Birmingham
Department College of Medical and Dental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We produced expression constructs and test purification of claudin proteins from E coli and mammalian cells.
Collaborator Contribution We have obtained access to inhibitors, high throughput crystallization and ligand screening methods, and have submitted joint grant applications.Provision of claudin constructs, ligands and binding assays; development of antibodies
Impact A PhD studentship has been recruited to further develop this new collaboration and set of claudin targets, which are structurally and functionally related to the original tetraspanin targets.
Start Year 2009
 
Title Licensing, collaboration and material transfer agreements 
Description The SMALP nanoparticle method to solibilise actve membrane proteins without resorting to destabilising detergents has been licensed and made available to >10 biotech, chemical and pharma partners. 
IP Reference WO2011004158 
Protection Patent application published
Year Protection Granted 2014
Licensed Yes
Impact Follow-on funding has been awarded from the BBSRC to further commercialise the SMALP system.
 
Title SMALP system 
Description A revolutionary new protein stabilisation technique has been developed which could lead to 30 per cent more proteins being available as potential targets for drug development - opening up exciting possibilities in drug discovery. Understanding the structure of proteins is a vital first step in developing new drugs, but to date, drug development has been slowed because due to their instability, proteins are difficult to work with in lab conditions. However, using SMALP nanoparticles we have found a way to preserve membrane proteins intact, enabling detailed analysis of their structure and molecular functions. 
IP Reference US2012142861 
Protection Patent granted
Year Protection Granted 2012
Licensed Yes
Impact Press release was issued that was reported on by 20 magazines worldwide. Collaborations have been established with ten pharmaceutical, biotechnology and manufacturing companies to develop new applications for our technology.
 
Title styrene maleic acid lipid particle (SMALP) system 
Description Membrane proteins are commercially the most valuable class of drug target as more than half of all marketed drugs target them. However, they are seriously underexploited due to the difficulties in studying them. We have developed the SMALP system to solubilise and stabilise membrane proteins including the most valuable drug targets in minutes and without any detergents. The SMALP system is a simple generic and inexpensive reagent that can be used for the preparation of pharmaceutical drug targets that yields significant improvements to their purification, stability and longevity over all conventional systems. The system is compatible with standard biophysical techniques and drug screening strategies. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact We have established collaboration agreements with 10 pharma and chemical companies to exploit the SMALP technology for any protein from any cell type, and plan to initiate commercialisation of the SMALP system in the next year. 
 
Company Name Science Capital 
Description Science Capital is a company limited by guarantee that I founded and direct. This nonprofit brings together scientists and business experts, through engaging talks and lively discussions, exploring scientific and technological advances alongside the investment and legal frameworks needed to make them a commercial reality. Topics encompass innovative healthcare and biomedical technologies, see www.sciencecapital.co.uk 
Year Established 2010 
Impact Science Capital has run quarterly meetings several times that have attracted hundreds of scientists and business leaders to forge new partnerships and initiate new business propositions.
Website http://www.sciencecapital.co.uk/
 
Description 23rd ICMRBS Conference, San Diego, USA 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact A poster was presented to the audience which includes the cream of international NMR community. The poster highlighted the problems in generating a eukaryotic membrane protein sample for structural studies and ways to overcome this. The feedback and discussions prompted us to make a few important modifications in our methods to address certain issues.

Collaborative interactions with companies and academic research groups were developed, and plans for an EU grant application on tetraspanins were initiated.
Year(s) Of Engagement Activity 2008
 
Description 2nd European Mini Symposium on Tetraspanins 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A poster describing some preliminary data and expression, purification and functional analyses of tetraspanins was presented. A new collaboration emerged from this meeting and is being pursued for funding.

Collabortive interactions were discussed and plans for a EU project applications were developed, including identification of suitable partners.
Year(s) Of Engagement Activity 2008
 
Description 3rd European Minisymposium on Tetraspanins 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact There were over 100 participants from senior academics to students and people from industry. The research presented generated keen interest in the production of tetraspanin proteins for structural analyses and preliminary collaborations have been ongoing with research groups in France.

Members of tetraspanin research community across Europe have requested expression and purification protocols that were used in the MRC funded project.
Year(s) Of Engagement Activity 2009
 
Description 4th European Minisymposium on Tetraspanins 
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 Over 120 participants from research groups across Europe, Japan and USA took part in this meeting. The tetraspanin expression system developed has generated keen interest for the development of powerful antibodies to be used as research tools.

Preliminary discussions for a collaborative EU project started with a group at the University of Mainz, Germany to explore tetraspanin signalling in HPV infection.
Year(s) Of Engagement Activity 2010
 
Description Annual Lecture of the Lunar Society 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Annual Lecture of the Lunar Society was given to 180 members of the public at the ThinkTank in Birmingham. Michael Overduin hosted and introduced this event (and about 3 other events each year for this organization.)

Press release on the events are regularly reported on in Birmingham newspapers, with a full two page article on the Annual Lecture entitled After Darwin's Watch appearing in the Post on 20 March 2009.
Year(s) Of Engagement Activity 2009
 
Description British Science Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Michael Overduin organised four events for the British Science Festival, which attracted over 80,000 attendees to 280 events, and served on its Steering Committee. The events were offered at the NMR facility and cetnral Birmingham venues.

Follow up tours of the NMR facility have been requested, and an ongoing series of events are being planned for continued engagement of the local business community involved in and supporting R&D and innovation.
Year(s) Of Engagement Activity 2010
 
Description Japan Partnering Award Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Sundaresan Rajesh and Michael Overduin organized and hosted this meeting for 25 scientists including members of pharmaceutical companies. A series or presentations and a training workshop were here to discuss the production and analysis of challenging proteins including tetraspanins.

Loan of a Protemist DTII robot to synthesize proteins by UK-based research groups.
Year(s) Of Engagement Activity 2008
 
Description Keystone Symposium 
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 M Overduin; M Jamshad; Y Lin; T Knowles; A Jazayeri; D Poyner; R Bill; M Wheatley; T Dafforn, Amphipathic polymer-based nanoparticles for purifying and characterizing stable and active GPCRs without detergents

collaborations were established to demonstrate methods for partners and initiate new collaboration with academic and industrial (pharmaceutical co.) partners
Year(s) Of Engagement Activity 2010
 
Description Parliament Magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact A full page article on our innovative research was prepared, submitted and published in the Parliament Magazine's Health Issue in Autumn 2009.

Further interviews were given and articles written for public awareness of our research.
Year(s) Of Engagement Activity 2009
 
Description Protein Island Matsuyama International Symposium 
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 200 people attended a talk about the applications of the research on membrane proteins, which initiated discussions, questions, and potential collaborations.

A followup meeting was planned and run a year later for the participants, and strong links between the UK and Japan were forged including development of new IP.
Year(s) Of Engagement Activity 2009
 
Description Public Service Review 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Michael Overduin and Sundaresan Rajesh submitted a two page article on our research in an easily accessible language and with colourful graphics, this appeared in the Public Service Review magazine in Nov 2009.

Further articles and interviews were invited and given.
Year(s) Of Engagement Activity 2009
 
Description Tour - Lord Mayor of Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact Michael Overduin gave several guided tours of the HWB-NMR research facility he directs at the University of Birmingham to several groups including one by the Lord Mayor on 8 Apr 2008.

The profile of the University of Birmingham was enhanced within the city and region.
Year(s) Of Engagement Activity 2008