On an epic scale

Studying proteins in Scotland

Two Scottish universities – Dundee and Edinburgh – won large awards through the Joint Insfrastucture Fund. Both bids were for equipment to enhance their already internationally renowned research programmes into the interactions, structures and functions of proteins.

The Universities of Dundee and Edinburgh have won numerous plaudits for their research on proteins in many different fields. Intent on remaining internationally competitive, they applied for the latest cutting-edge equipment, such as that for proteomics and for nuclear magnetic resonance (NMR) studies, through the Joint Infrastructure Fund (JIF). Both bids were successful: the University of Dundee was awarded £3.6 million to establish a Post-Genomics and Molecular Interactions Centre; and the University of Edinburgh was awarded £4.7 million to establish EPIC, the Edinburgh Protein Interaction Centre.

Both facilities will provide important central resources for researchers from a number of departments. In Dundee, the JIF application (led by Professor Mike Ferguson) was for equipment for proteomics, bioinformatics, DNA sequencing and synthesis, structural analysis (NMR spectroscopy) and analysis of molecular interactions. This rich array of equipment will be in demand by a host of leading scientists working on a diverse range of molecular and cellular systems – from the structure and synthesis of parasite coat proteins to the structure of DNA and RNA and their interactions with proteins.

The Edinburgh application, led by Professor Peter Sadler, has a strong component of chemistry applied to biology and again will be a widely used central resource. EPIC will include facilities for, among other things, protein purification and sequencing, structural biology (NMR spectroscopy) and combinatorial chemical synthesis. The study of enzymes will be a particular focus, but again many groups will be able to make great use of the facilities – from studies of splicing to work on the protein complexes causing neurodegenerative disorders such as Creutzfeldt–Jakob disease and Alzheimer's disease.

Here, four researchers discuss the impact the new technology will have on their studies – illustrating how the study of many biological questions can be accelerated by new and enhanced methods for analysing proteins and their interactions.

On the surface

Professor Mike Ferguson, director of the nascent proteomics suite at the University of Dundee, is investigating the cell-surface molecules of parasites.

The trypanosome parasites cause sleeping sickness in Africa and Chagas disease in South America, while the related Leishmania parasites cause a variety of diseases in many different parts of the world. "These parasites are all related, so we are studying them as a group," says Professor Ferguson. "We are interested in the form and function of the cell-surface molecules of these organisms, and how these molecules enable the parasite to survive and replicate in the host."

Over many years, Professor Ferguson's group has been isolating various surface components and determining their chemical structure. All of the major surface molecules on these organisms – whether glycoproteins (proteins with sugars attached) or glycolipids (sugars and fats) – belong to one common family known as the GPI (glycosylphosphatidylinositol) family, the GPI being the final anchoring unit that attaches the molecules to the cell surface. "This family of molecules is found across all these related parasites," says Professor Ferguson, "and this is true of other parasites such as malaria. Even though the shapes of these cell-surface molecules are very different between the parasites, reflecting their different lifestyles and life cycles, all of the major molecules that are required for their infectivity are anchored in the same way to the plasma membrane."

"So, having come to that realisation, we are focusing on the biosynthesis of this GPI unit and trying to develop drugs that will interfere with the unit's assembly. If we are successful in that, we may have a pan-specific drug against all the parasites. We’ve been doing that quite successfully by chemically synthesising various GPI structures to use as inhibitors, and moving steadily towards lead compounds as potential therapeutic agents."

Professor Ferguson's group has been using mass spectrometry routinely for these structural analyses. As proteomics is, at present, predominantly a mass spectrometry-based technology, it was a natural progression for them to take proteomics into their fold and to get it started up in Dundee. "We use proteomics to identify new components of the biosynthetic pathway that associate with the components we have isolated previously," says Professor Ferguson. "With this technology you need only a tiny amount of a new protein to go straight to the gene that encoded it in the first place. It's an incredibly rapid process. Previously, if you were interested in a component of the pathway, you had to go through rigorous biochemical purifications, following a biochemical activity. This is almost impossible for some components of the GPI pathway because they are associated with the cell membrane and lose all activity as soon as you solubilise the membrane."

"We've done it the hard way in the past," says Professor Ferguson. "In one case, we purified an enzyme of the pathway, got the peptide sequence and cloned the gene; it took six years. Now, with proteomics, if we get hold of one component we stand a very good chance of getting the other components simply by 'guilt by association'. It takes only about six months."

Professor Mike Ferguson holds a Wellcome Trust programme grant.

The watchmen

Professor Colin Watts, also in Dundee, is investigating how dendritic cells – the body's 'look-outs' for infection – capture foreign material and spark an immune response.

Scattered around the body's tissues, dendritic cells act as sentinels, keeping a look-out for foreign invaders. If there is a bacterial or viral infection, the dendritic cells capture some of the products made by the pathogens, become activated and migrate to the draining lymph nodes. There, they 'present' the antigen to the T cells, initiating an immune response. Dendritic cells are able to sense and sample the local environment and respond to pathogen products – such as bacterial cell wall material and viral double-stranded DNA and many others – by launching a program of maturation and migration. At the same time protein antigens from these pathogens are captured, processed and presented on major histocompatibility complex (MHC) molecules. Professor Watts is particularly interested in the changes that take place in the antigen capture and presentation machinery upon maturation.

While in the tissues, the dendritic cells are immature, concentrating on taking up antigen by endocytosis. Capture of a foreign antigen sparks a programme of maturation, during which the cells shut down endocytosis and turn their attention to processing the antigen and presenting it in association with MHC molecules: "the currency that T cells understand," as Professor Watts describes it. "If we can understand how the system of capture, processing and presentation works, we may be able to make vaccines more effective, or create new opportunities to intervene when the immune system is switched on inappropriately – such as in allergy, rejection of transplants or autoimmunity."

"One of the things we want to do with proteomics is to look, at the protein level, at some of the changes that take place in the dendritic cells during the process of maturation," says Professor Watts. "In particular, we are looking at changes in the machinery that controls the cytoskeleton, the internal scaffolding of the cell. In the tissues, the cell is using the cytoskeleton to drive endocytosis. Maturation shuts down most endocytotic activity and the cytoskeleton becomes important in other processes such as long-distance migration and interactions with T cells."

Likely candidates for change during maturation are proteins that interact with the Rho family of GTPases. The Rho proteins themselves switch numerous processes in the cell on or off, and are known to control changes in the cytoskeleton, but Professor Watts and colleagues have already found that their activity does not alter significantly during maturation. "So what may change is the proteins with which the GTPases interact, and we can use proteomics to look at the interactions in immature and mature dendritic cells."

"These new facilities will allow us to do experiments that, frankly, would be very difficult to do otherwise," says Professor Watts. "It will be much better to have this facility in-house, so that we can just go downstairs and do the experiments interactively with the people running the proteomics set-up here in Dundee. We're very hopeful that we can find new possibilities for manipulating cells to improve the uptake of vaccines or to optimise the overall process of the presentation of vaccines to the immune system."

Professor Colin Watts holds a Wellcome Trust programme grant.

Your flexible friends

In Edinburgh, Paul Barlow is taking a nuclear magnetic resonance approach to deduce the structures of the big, flexible proteins that regulate the immune system's first line of defence against infection – complement.

The structures of protein domains or 'folds', stable three-dimensional structures that often make up a significant proportion of a protein's overall structure, can provide important insights into how a protein functions. The linkers between domains are often less defined structures, but this lack of regular structure does not mean that these regions are unworthy of scrutiny. "My interest is in proteins that have multiple domains, but not so much any longer in the domains themselves, but in how these domains are organised into a functional whole protein," says Dr Barlow. "In fact, many proteins are not assembled into a defined three-dimensional structure, and are quite flexible or floppy. This flexibility can be an intrinsic part of their function as they need to be able to form large complementary surfaces with their targets. At the same time they don't necessarily want to bind particularly tightly – so they flop on to their target, hold on gently for a while and then flop off."

As the flexibility of these proteins makes them difficult to study, Dr Barlow uses nuclear magnetic resonance (NMR) in his research, a particularly useful technique for such problems as the structures of proteins are determined while they are in solution (rather than being crystallised into a fixed shape). Through JIF, the Edinburgh Protein Interaction Centre will be getting a new 800 MHz NMR machine. "This new machine will provide better sensitivity and resolution than our existing instruments," says Dr Barlow. "It will also allow us to examine larger proteins, often a limitation of NMR."

Together with collaborators in the USA, Sweden and Australia, Dr Barlow and colleagues are examining proteins that regulate the immune complement system – the first line of defence against invaders such as bacteria, viruses, fungi and parasites. Complement is somewhat error-prone, however, being involved in an enormous range of pathologies, from Alzheimer's disease, burns and Crohn's disease to transplant rejection.

"We are interested, ultimately, in finding therapeutics that can dampen down the complement system when it is operating inappropriately," says Dr Barlow. "So we are looking at a family of multiple domain proteins that are the natural regulators of complement: factor H has 20 similar domains, separated by short linkers, and complement receptor 1 has 30 domains. As you might imagine, both these proteins are big, extended, and floppy. An engineered version of complement receptor 1 is already being used in the clinic – in transplant surgery – to inhibit complement reactions. But small molecule therapeutics that can interact with the natural regulators of complement would be even more useful."

A viral protein whose structure has been solved by Dr Barlow and colleagues may provide important clues for the design of such inhibitors. "The poxvirus has a four-domain protein closely related to factor H and complement receptor 1," says Dr Barlow. "The virus has presumably pirated the gene from humans or another mammal. We can learn a lot from this protein, as it is used by the virus to protect itself against complement, and it’s a really good inhibitor. We need to find out exactly how it clings onto its target."

Paul Barlow holds several grants from the Wellcome Trust (and other funding agencies).

Slicing and dicing

Edinburgh-based researchers Professor David Tollervey and colleagues have used proteomics to identify the exosome – a complex of proteins that trim and chop RNA.

Every cell contains a multitude of protein-synthesis factories, the tiny particles called ribosomes. Each ribosome is made up of RNA (about 60 per cent) and protein (about 40 per cent), with four different RNA species and about 70 different proteins coming together to form the final complex. "The cell produces huge amounts of ribosomal RNA (rRNA)," says Professor Tollervey. "These are made as long precursor RNAs, which are processed and trimmed to get the mature RNA ready for use in the ribosome. If one of the enzymes responsible for processing rRNA is faulty, the synthesis of the ribosomes themselves is defective and the cells die as they cannot make new protein."

The enzymes responsible for processing RNA are termed exonucleases or endonucleases, as they cut the RNA at the ends or the middle, respectively. "We had identified a mutation in a gene that caused yeast cells to be defective in the synthesis of ribosomes," says Professor Tollervey, "and found out that the gene encoded an exonuclease that trims the ends of an rRNA."

Having tagged the exonuclease protein, they looked for proteins that it associated with – the proteins being identified using mass spectrometry by Matthias Mann's group in Germany (now in Odense, Denmark). "We have identified 12 proteins so far – although only 11 of these proteins are in a complex at once," says Professor Tollervey. "Proteomics was absolutely key to identifying these proteins: some of the components had been identified by other methods, but the only way we knew they were in the same complex was by co-purifying them and identifying them by mass spec."

Two forms of the complex have been identified – one in the nucleus and one in cytoplasm – which have 10 common components and an extra protein specific to each form. "The complex is called the exosome because nine of the ten common components have been shown to be exonucleases or are strongly predicted to be exonucleases," says Professor Tollervey. "The cytoplasmic exosome is involved in the turnover of messenger RNA (mRNA), while the version in the nucleus is involved in a whole host of processing reactions: nuclear pre-mRNA, rRNA, small nuclear RNAs and small nucleolar RNAs."

Professor Tollervey is already planning a plethora of new experiments to take advantage of the new proteomics equipment coming to Edinburgh. "We want to use the same approach to look at other complexes, in particular how ribosomes are assembled and built into fully functional complexes. MALDI is by far the quickest and easiest way to do many of our experiments – for the exosome in particular because six of the proteins are very similar in size. We could identify them without a mass spec, but it makes life a lot easier to find out which one is which."

Professor David Tollervey is a Wellcome Trust Principal Research Fellow.

External links

Dundee Biocentre at the University of Dundee
Professor Mike Ferguson : Director of the proteomics suite at the University of Dundee
Professor Colin Watts at the University of Dundee investigates immune responses in dendritic cells
Institute of Cell and Molecular Biology , University of Edinburgh
EPIC : Edinburgh Protein Interaction Centre
Professor David Tollervey uses proteomics to identify the exosome
Dr Paul Barlow uses NMR to deduce the structures of proteins
Matthias Mann : based at the University of Southern Denmark