Biochemical analysis of human IQGAP proteins

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

In your cells there is a network of proteins called the cytoskeleton. This isn't a very good name because unlike your bones it is constantly being taken apart, put back together and moved around. Its job is to provide support for the cell, to provide tracks for things to move about in the cell on and to help the cell move around. All these activities need to be co-ordinated and if this goes wrong you'll get ill. You might even get cancer. One of the most interesting recent discoveries is that there are a group of proteins whose job it is to help organise the cytoskeleton. These organising proteins (called scaffolding proteins) do more than just bring the right proteins together at the right time. They also talk to other proteins by sticking to them. When two proteins stick to each other messages can be passed between them because of the different shapes the molecules can adopt. So we think these scaffolding proteins listen to what other proteins have to say, work out what it all means and then pass the right message on to the cytoskeleton. You can think of them being like miniature telephone exchanges. We want to know how they work. The particular scaffolding proteins we are interested in are called the IQGAP proteins. In your body you have three different types of IQGAP protein called IQGAP1, IQGAP2 and IQGAP3. Don't worry / the name IQGAP doesn't mean that you're stupid because you have a gap in your IQ. It's actually named after two parts of the protein / the IQ-motifs and the GAP domain. These are two of the bits of the protein which stick to other proteins. The IQ-motifs stick to a protein called calmodulin and the GAP domain to one called CDC42. Both calmodulin and CDC42 are proteins which carry messages round the cell. There's another bit we're interested in called the CHD which sticks to actin. Actin is a protein in the cytoskeleton. So we think IQGAPs collect information from calmodulin and CDC42 and pass it on to actin. We want to find out how they do that. What we're going do is to genetically engineer some bacteria so that they'll make calmodulin and CDC42 for us. IQGAPs are a bit big to do this with and so we'll make fragments of them. Luckily for us, other scientists have already worked out some fragments which bacteria are happy to make. We've also got a problem with actin. Other people have shown that when bacteria make this protein it doesn't work properly. So we'll make that one in yeast instead. Once we've got all these proteins we'll ask how they stick together. We'll try and find out the chemical details of what's going on at the places where the proteins stick together. Of course proteins are very very small and so we can't just look at them. We can't even look at them under a microscope. So we have to use indirect methods using chemistry and physics to find out what's going on. We're also going to find out how tightly they stick / again using chemical and physical methods. Another thing we're very interested in is how IQGAP1, IQGAP2 and IQGAP3 differ from each other. We know that they are quite similar, but we also know that IQGAP1 and IQGAP2 are found in different cells and stick to a slightly different range of other proteins. As for IQGAP3 we really don't know anything about that yet. So anything we discover about it really will be novel. We think that the way each IQGAP sticks to these other proteins will be slightly different and that how strongly they stick will also be different. Hopefully once we know the chemical details about how they stick, we'll also have a good idea about why the strength of stickiness is different. This project is important because the cytoskeleton goes wrong in a lot of diseases and some drugs interact with the cytoskeleton. When we've finished we'll be a bit closer to understanding how these tiny molecular telephone exchanges actually work.

Technical Summary

This project will begin the process of understanding the biochemical details of how a key group of cytoskeletal scaffolding proteins, the human IQGAPs, work at a molecular level. This will be tackled by initiating in vitro studies of the three human IQGAP proteins (IQGAP1, IQGAP2 and IQGAP3) using a compare and contrast approach. Fragments of these proteins along with four key partners (calmodulin, myosin essential light chain, actin and CDC42) will be expressed in yeast or bacteria and purified to homogeneity. The interfaces between the IQGAPs and their binding partners will be mapped using chemical cross-linking and protease protection. The stoichiometries of binding will be determined by analytical gel filtration and the affinities by surface plasmon resonance (BIAcore) analysis.

Publications

10 25 50
 
Description We have worked out some details of how a "molecular telephone exchange" called IQGAP works. We hope that this will be useful in the development of new drugs and new devices.
Exploitation Route They provide insight into the biochemical mechanisms of IQGAP1,2 and 3. Isoforms 2 and 3 remain relatively understudied and our work on these (calmodulin and myosin light chain binding) will be useful to other researchers.

The award catalysed two current external and one internal collaboration. The external collaborations involve the exchange of material (proteins, expression plasmids) to facilitate biochemical, biophysical and molecular cell biological studies on IQGAP1. It is anticipated that at least one (hopefully both) of these will lead to further publications in this area. The internal collaboration involves the provision of material (proteins) for testing of a novel biosensor. This is being developed by colleagues at QUB and through a spin-out company, Causeway Biosensors.

There is now considerable interest in the role of IQGAPs in cancer. Of particular relevance to this award, is the growing realisation that key interactions may be critical in initiating or maintaining the transformed state in cells. Therefore, targeting these interactions pharmacologically may enable the design of novel anti-cancer drugs. Two grant applications in this field were submitted but unfortunately neither were successful. It is possible that the potential for funding on this topic will be greater after the collaborative work with external groups is completed and published.
Sectors Retail

 
Description They have been exploited and cited by other research groups globally. It is possible that some exploitation is occurring in pharma/biotech industries but we would not expect to see any public outputs (eg new drugs, patents etc) for some time (possible another decade).
First Year Of Impact 2007
 
Description PhD studentship
Amount £65,000 (GBP)
Organisation Government of Northern Ireland 
Department Department for Employment and Learning Northern Ireland (DELNI)
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2011
 
Description Summer studentship
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2009 
End 09/2009
 
Description Wellcome Trust Summer Studentship
Amount £1,800 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2010 
End 09/2010
 
Title Expression plasmids for IQGAP regions and IQGAP binding partners 
Description The following IQGAP expression constructs were made as part of this project: Calponin homology domain from human IQGAP1, IQGAP2 and IQGAP3 RasGAP C-terminal domain (RGD) from IQGAP1 Short C-terminal region (minus RGD) from IQGAP1 and IQGAP3 IQ-motifs region from human IQGAP1 (various fragments) and Saccharomyces cerevisiae Iqg1p (various fragments) The following human binding partner expression constructs were made: Calmodulin Myosin light chain Mlc1sa S100b CDC42 Rac1 APC (various fragments) The following Saccharomyces cerevisiae binding partner expression constructs were made: Mlc1p Cmd1p 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2010 
Provided To Others? Yes  
Impact Other groups are using them in their research; we await publications 
 
Description Dusseldorf 
Organisation Heinrich Heine University Düsseldorf
Department Institute of Biochemistry and Molecular Biology II
Country Germany 
Sector Academic/University 
PI Contribution Supply of plasmids and proteins (for the tractable fragments of IQGAP1 developed in the project)
Collaborator Contribution Biophysical experiments on IQGAP1 and its interactions
Impact None yet; experiment underway
Start Year 2010
 
Description Jaffna 
Organisation University of Jaffna
Country Sri Lanka 
Sector Academic/University 
PI Contribution Ideas and research advice on yeast IQGAPs
Collaborator Contribution Some computational work; student nominated for Sri Lankan President's award (PhD studentship) - outcome not yet known - to come and study at QUB
Impact MSc project at University of Jaffna. Medium term potential for publication.
Start Year 2010
 
Description Texas 
Organisation University of Texas
Department Health Science Center at Houston
Country United States 
Sector Academic/University 
PI Contribution Supply of proteins (other researchers are interested in our fragments of IQGAP1 since they are more biochemically tractable than some from other groups).
Collaborator Contribution Experiments on the role of IQGAP1 in a signalling system not previously linked to IQGAP1
Impact None yet; experiments still underway.
Start Year 2012
 
Description Workshop for school children 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact talk sparked questions and discussion afterwards

Increased engagement with local schools and teachers; several current students at QUB have mentioned the days as something which inspired them to study science at University
Year(s) Of Engagement Activity 2007,2008